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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/10—Protozoa; Culture media therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/10—Protozoa; Culture media therefor
  • C12N1/105—Protozoal isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/90—Protozoa ; Processes using protozoa

Definitions

• the present invention relates to the use of attenuated live parasite strains for the prevention and / or treatment of ocular lesions associated with Toxoplasma gondii infection, in mammals and in humans in particular.
• Apicomplexes are obligate intracellular protozoan parasites that have a life cycle that can involve multiple hosts.
• the phylum of these parasites is subdivided into several families.
• Toxoplasma gondii belongs to the family Sarcocystidae. This protozoan exists in three infectious forms that vary according to the host and the infectious stage:
• tachyzoite a proliferative and infectious form that multiplies asexually in the cells of the intermediate hosts (i.e. all the homeotherms) and the final ones (i.e. the felids and the cat in particular),
• o bradyzoite a slow-dividing, low-level form of metabolism of the parasite contained in cysts
• sporozoite a form contained in oocysts, which results from the sexual multiplication of the parasite in the intestine of definitive hosts (i.e. cat and other felids).
• the cat the definitive host of the parasite, becomes infected by ingesting parasitized prey containing cysts (or tachyzoites if the prey is in the acute phase of toxoplasmosis (Dubey, 2002, J. Parasitol, 88: 713-717)), or by ingestion of oocysts. Infection of the cat leads to the formation, in its gut, of gametocytes whose fusion leads to the formation of oocysts which will then be disseminated in the environment via feces. These oocysts, which contain the sporozoites, sporulate and remain very long infectious in the external environment. Their pathogenic character persists for at least one year.
• the released sporozoites After ingestion of oocysts by the definitive and intermediate hosts, the released sporozoites infect the enterocytes of the host and become tachyzoites that are disseminated in the body. Under the pressure of the immune system, the Tachyzoite is transformed into bradyzoite which persists in cysts with a preferential tropism for the central nervous system, retina and muscles.
• bradyzoites are released and infect the entero-epithelial cells and turn into tachyzoites, which disseminate in the host organism and, under the pressure of the immune system, will again form bradyzoites intra-cystic.
• T. gondii strains are classified into three types (Howe et al, 1995, J.
• Type I strains i.e. strain RH
• Type II strains ie, strains ME49, 76K or
• Prugniaud and III are relatively less virulent and generally establish chronic infections (Howe et al., 1997, J. Clin.
• Toxoplasma gondii causes an infectious disease: toxoplasmosis. Extremely widespread, with more than one-third of the world's population infected, the consequences of toxoplasmosis can be dramatic in two particular cases: (i) in HIV-negative pregnant women since the parasite can cross the placental barrier and infect the fetus thereby causing an abortion or severe malformations or severe psychomotor disturbances in the newborn and (ii) in immunocompromised persons such as HIV-infected persons in whom toxoplasmosis, an opportunistic parasitosis, can cause serious brain or heart disorders, lethal in the absence of treatment.
• toxoplasmosis In immunocompetent persons, toxoplasmosis is usually benign. However, in recent years, many publications describe the impact of this infectious disease on the eye. The retina constitutes a preferential site of encystment of the parasite. Thus, toxoplasmosis is the most common cause of intraocular inflammation and posterior uveitis in individuals immunocompetent. These intraocular inflammations can cause a modification of the constituents of the ocular fluids (aqueous humor and vitreous humor) and thus alter the visual function. Toxoplasmosis is also responsible for retinochoroiditis most often related to the reactivation of parasites contained in retinal cysts, cysts may result:
• T. gondii a congenital infection
• Infection of the fetus in the first weeks of pregnancy leads to major ocular lesions (microphthalmia, cataract, extensive foci of retinochoroiditis).
• Ophthalmological monitoring of children infected with T. gondii in utero should be performed regularly during the first seven years of life of the child.
• Follow-up of functional signs ⁇ i.e. impaired vision, myodesopsia, scotoma) should immediately direct the practitioner towards a reactivation of T. gondii characterized by a multiplying phase of the parasite, o either of an acquired infection.
• Congenital infection is not the only etiology of ocular toxoplasmosis. Indeed, the role of infections of acquired origin has been demonstrated in many cases of ocular toxoplasmosis, especially in several members of the same siblings or when the prevalence is high in certain geographical areas. In ocular toxoplasmosis of acquired origin, ocular manifestations may be concomitant to primary infection or delayed, sometimes years or decades after primary infection.
• T. gondii resulting from an acquired infection is comparable to that resulting from a congenital infection and regardless of their etiological origin, the lesions generated on the retina may cause deterioration or loss of vision if they are close to the macula or optic nerve.
• toxoplasmic retinochoroidal scars were found in 17.7% of the subjects examined and out of a total population of 2 million, 20,000 people lost their use of one eye and 5,000 people are totally blind (Jones et al, 2006, Emerg Infect Dis, 12: 582-587, Silveira et al., 2009, Expert Rev. Anti Infected Ther 7: 905-908).
• the diagnosis of ocular toxoplasmosis is essentially clinical.
• gondii can be performed not only in the patient's serum, but also in the aqueous humor of the eye. Measurements of anti-T. gondii IgG levels and total IgG levels in these two compartments make it possible to determine the value of the Desmonts coefficient. This coefficient corresponds to the ratio IgG anti-T. gondii I total IgG in the aqueous humor on the ratio IgG anti-7 7 . gondii I total IgG in the blood. When this ratio is greater than 3, it is estimated that there is a local synthesis of anti-7 7 antibodies.
• gondii translating intraocular infection.
• this ratio is less than 2, the local production of anti-7 7 antibodies. gondii is not demonstrated, without ocular toxoplasmosis being eliminated.
• a value between 2 and 3 is doubtful for asserting local production of T. gondii antibodies.
• the presence of the parasite in the aqueous humor can be detected by the amplification of T. gondii DNA by the Polymerase Chain Reaction technique.
• Atovaquone has the best in vivo action potential on both tachyzoites and bradyzoites. These compounds must be used at high concentrations to cross the blood-eye barrier and reach sufficient levels in the retina. From this In fact, these drugs can have dramatic side effects (Lyell's syndrome, agranulocytosis, pseudo-membranous colitis) and require supplementation of the patient with folate.
• the use of these compounds may be associated with corticosteroid therapy.
• the goal of corticosteroids is to limit the inflammatory reaction associated with retinochoroiditis of toxoplasmic origin. Prednisone is the reference corticosteroid. Administration of corticosteroids, however, is reserved for immunocompetent patients.
• T. gondii an attenuated live vaccine strain of T. gondii has been developed by the invalidation of two genes encoding the TgMIC1 and TgMIC3 proteins (EP 1 703 914 B1 and US 7,964,185 B2 / Cérrow et al., 2005 J. Exp Med, 201: 453-63).
• This strain called Toxo micl-3 KO, generates a strong and specific immune response against T. gondii and makes it possible to prevent the effects of a subsequent infection in mice (Isma ⁇ l et al, 2005, J. Infect Dis., 194 1176-1183) and also in ewes (Mevelec et al., 2010, Vet Res., 41: 49-60).
• One of the aims of the invention is to provide an agent for reducing, in the eye, in a mammal, the inflammation following a T. gondii infection.
• Another object of the invention is to reduce the inflammatory reaction occurring in the eye at the time of reactivation of T. gondii in a mammal already infected with T. gondii.
• Another object of the invention is to provide an agent for reducing the number of cysts, especially intra-retinal, thereby preventing eye lesions related to the reactivation of T. gondii.
• Yet another object of the invention is to provide a vaccine against ocular lesions caused by T. gondii.
• the subject of the present invention is strains of Toxoplasma gondii isolated from their natural environment for use in the prevention and / or treatment, in a mammal, of ocular lesions associated with an apicomplex infection of the family Sarcocystidae.
• the subject of the present invention is strains of Toxoplasma gondii isolated from their natural environment for use in the prevention and / or treatment, in a mammal, of ocular lesions associated with infection by an apicomplex of the family Sarcocystidae, said strains possessing attenuated virulence with respect to a virulent T.gondii RH strain.
• the subject of the present invention is strains of Toxoplasma gondii isolated from their natural environment for use in the prevention or treatment, in a mammal, of ocular lesions associated with an apicomplex infection of the family Sarcocystidae, said strains having a attenuated virulence with respect to a virulent strain, i.e. a strain having a virulence substantially identical to the virulence of the strain from which the attenuated virulence strain was obtained.
• "Prevention” refers to prophylaxis aimed at preventing the onset or spread of a disease. These include protecting an individual predisposed to the contraction and development of ocular lesions associated with an apicomplex infection of the family Sarcocystidae. This includes protecting a mammal exposed to a risk of contamination by its environment.
• treatment is meant not only the inhibition of the progression of the pathology, but also the attenuation, or even the disappearance, of the symptoms related to this pathology.
• the aim of the treatment is to reduce the amplitude of the symptoms until their complete disappearance, allowing the individual to return to a normal physiological state.
• “Mammal” refers to humans, pets and livestock or livestock, which are of economic and commercial value to the agri-food industries.
• strains of Toxoplasma gondii is meant strains of Toxoplasma gondii which have an attenuated virulence, less than the virulence of RH-type T.gondii strains from which they derive, but which nonetheless retain an immunogenic capacity identical to that of the strains.
• T. gondii type RH for use in the prevention or treatment of a pathology associated with an apicomplex infection of the Sarcocystidae family. This attenuated virulence can result from the invalidation of at least one gene linked to the virulence of the parasite.
• This gene invalidation can occur during a natural process of evolution of the species or be performed in vitro by molecular biology techniques well known to those skilled in the art.
• the gene invalidation occurs at random in the genome whereas in the other case, the gene invalidation is targeted at one or more specific genes. Whether of natural origin or consecutive to the human hand, this gene invalidation results in the absence of expression of the protein encoded by the invalidated gene (s) or the expression of one or several non-functional proteins.
• the in vitro modification of the genetic heritage of Toxoplasma gondii confers on the strain a mutant character, as opposed to the wild strain from which it derives. Wild strains of parasites not only have an immunogenic potential but are also virulent, i.e. they are capable of inducing pathology associated with Toxoplasma gondii (ie toxoplasmosis), rendering their use unsuitable in the context of the present invention.
• Pathologies associated with apicomplex infection of the family Sarcocystidae means diseases resulting from infection with a protozoan belonging to the phylum apicomplexes, and in particular parasites belonging to the family of sarcocystidae which includes the genus Toxoplasma.
• strains of Toxoplasma gondii isolated from their natural environment said mammal is a human being or an animal.
• said strains of Toxoplasma gondii isolated from their natural environment said strains of Toxoplasma gondii have at least one adhesin MIC-1 and / or an adhesin MIC-3 inactivated by a gene modification involving at least one of the mic-1 and / or mic-3 genes.
• strains of Toxoplasma gondii isolated from their natural environment have at least one adhesin MIC-1 and / or an adhesin MIC-3 inactivated by the deletion of at least one of the mic-1 and / or mic-3 genes.
• a MIC-1 adhesin and / or an adhesin MIC-3 is meant the proteins of the micronemes, also called adhesins, MIC-1 and / or MIC-3, which play a role in mobility, migration or migration. cellular invasion of the phylum of the apicomplexes in its host. These proteins have binding modules that allow them to bind to host cells.
• an inactivated adhesin is meant an adhesin whose function can no longer be provided within the cell.
• An adhesin is inactivated when it is not produced or when it is produced but has no functional activity.
• the inactivation may also be the consequence of inoperative or insufficient post-translational modifications (ie glycosylation, isoprenylation, phosphorylation, sulfation, amidation, acetylation, alkylation) of the adhesin which do not allow it to perform its function.
• Inactivation of an adhesin can also be obtained indirectly by altering or suppressing the expression of one or several other proteins (including other adhesins) that bind to the adhesin to form a functional complex. The destructuring of such a complex causes a loss of function of the adhesin.
• gene modification is meant any mutation made in the nucleic sequence of a gene resulting in the absence of expression of the protein encoded by this gene or resulting in the expression of a non-functional form of the encoded protein. by this gene. This operation requires the intervention of the hand of a person skilled in the art when it is operated in vitro.
• This mutation may consist in the deletion of all or part of the gene, or its coding region, or its promoter region, in the insertion or substitution of nucleotides in the nucleotide sequence of the gene.
• mic-1 gene is meant the gene coding for the MIC-1 microneme protein, also called MIC-1 adhesin. This protein contains several modules including binding domains that specifically bind lactose. The MIC-1 protein is also able to bind to the surface of the host cells.
• mic-1 gene is meant the gene encoding the micronemas protein
• MIC-3 also called MIC-3 adhesin.
• This protein is homodimerized to form a complex of 90 kDa.
• MIC-3 has EGF-like domains and a lectin-like domain.
• the MIC-3 protein is also able to bind to the surface of the host cells.
• strains of Toxoplasma gondii isolated from their natural environment said strains of Toxoplasma gondii possess the two adhesins MIC-1 and MIC-3 inactivated by a gene modification involving the two genes mic-l and mic-3.
• strains of Toxoplasma gondii isolated from their natural environment said strains of Toxoplasma gondii have both adhesins MIC-1 and MIC-3 inactivated by the deletion of both mic-1 and mic-3 genes.
• strain modification on the two mic-1 and mic-3 genes is meant the mutation made in the nucleic sequence of the mic-1 gene and that of the mic-3 gene. This double mutation results in the lack of expression of MIC-1 and MIC-3 proteins or results in the expression of a non-functional form of MIC-1 proteins. and MIC-3.
• This mutant strain of Toxoplasma gondii is called Toxo honey -3 KO and has a very attenuated virulence in comparison with the wild type T. gondii RH strains from which it derives.
• the Toxo micl-3 KO strains retain a high immunogenicity.
• the detailed construction of the strain Toxo micl-3 KO is described in documents Cérre et al, 2005 J. Exp. Med., 201: 453-63, US 7, 946, 185 B2 and EP 1 703 914 B1.
• Toxo micl-3 KO strains retained their ability to colonize the target tissues without the development of any pathogenic phenomenon following the administration of said strains to a mammal. Invalidation of the honey and mic3 genes in no way alters the immunogenic potential of these strains, but considerably reduces their virulence with respect to a virulent strain, that is to say a strain having a virulence substantially identical to virulence. of the strain from which the attenuated virulence strain was obtained.
• strains of Toxoplasma gondii isolated from their natural environment said apicomplex of the Sarcocystidae family is Toxoplasma gondii.
• Toxoplasma gondii refers to the protozoa of the phylum apicomplexes that can cause birth defects or miscarriages in most warm-blooded mammals and birds. These parasites are particularly capable of causing toxoplasmosis in humans, pigs, ewes, rodents or any mammal that has a mature or deficient immune system.
• strains of Toxoplasma gondii isolated from their natural environment said ocular lesions belong to the group comprising or consisting of intra- ocular inflammations, uveites, hyalites or retinochoro ⁇ dits.
• Intraocular inflammations any secretion of cytokines and chemokines involved in the immune response and any recruitment or activation of the immune system cells contained in the eye.
• Uveitis means the inflammation of the uvea, a vascularized complex that feeds the eye and includes the iris, the ciliary body (anatomical element to which are connected the ligaments retaining the lens) and the choroid. This inflammation can be caused by a virus, a bacterium or a parasite or be due to an autoimmune disease.
• Uveitis can be anterior, intermediate or posterior depending on the compartments of the eye it affects. Uveitis can be painful or not and is associated with a decrease in visual acuity. The eye may appear red with, for example, watery eyes or photophobia.
• hyalites inflammation of the vitreous of the eye which corresponds to an intermediate uveitis.
• retinochoroidites clinical entities that include any lesion of the retina-choroid complex. Always inflammatory, they can be of infectious or autoimmune origin. Retinochoroiditis therefore corresponds to an inflammation of the posterior uveae, that is to say of the retina and the choroid.
• strains of Toxoplasma gondii isolated from their natural environment said ocular lesions are consecutive to a primary infection of said mammal by T. gondii.
• primary infection is meant the first contact of said mammal with the parasite T. gondii. This primary infection leads to an immune response characterized in particular by the production of anti- ⁇ antibodies. gondii and by the activation of a cellular immune response directed specifically against T. gondii.
• strains of Toxoplasma gondii isolated from their natural environment said ocular lesions are consecutive to a reactivation of the asexual form of T. gondii.
• reactivation of the asexual form of T. gondii is meant the conversion of T. gondii bradyzoites to tachyzoites and the release of these after cyst disruption, thereby inducing cell contamination.
• the reactivation of T. gondii may be due to a deficiency of the immune system of the infected mammal.
• strains of Toxoplasma gondii isolated from their natural environment said ocular lesions are consecutive to a primary infection of said mammal by T. gondii and reactivation of the asexual form of T. gondii.
• strains of Toxoplasma gondii isolated from their natural environment said strains are contacted with said mammal at a rate of 100 to 10 8 tachyzoites.
• tachyzoite is meant the rapid and asexual form of replication of Toxoplasma gondii.
• the tachyzoite has a size of 5-8 x 2-3 ⁇ .
• the apical portion of the parasite has conoids that participate in parasite entry into the host cell.
• Micronemas, rhoptries and dense granules are the three main organelles of the tachyzoite, which also contains a nucleus, an apicoplast, a Golgi apparatus, an endoplasmic reticulum and an organelle close to the mitochondria.
• the effective dose of tachyzoites for the prophylactic treatment of mammals limits the infection or transmission of the pathogen responsible for toxoplasmosis.
• Another goal is to prevent the appearance of new intraocular lesions during reactivation of the encysted parasite.
• Such a treatment can be adapted and / or repeated as many times as necessary by those skilled in the art, depending on the age and the immunological status of the mammal.
• the strains of Toxoplasma gondii isolated from their natural environment for their use according to the present invention are in a dosage form chosen from the group comprising or consisting of liquid suspensions, solid or liquid dispersions, powders , pasta or lyophilisais.
• the dosage form is adapted by those skilled in the art depending on the mode of administration chosen. All the conventional modes of administration can be envisaged: enterally (per os for example) parenterally (intravenous, intramuscular or intraperitoneal injection for example) or by intra-nasal spraying.
• strains of Toxoplasma gondii isolated from their natural environment for their use according to the present invention may be associated with at least one other antigen, at least one adjuvant, at least one stabilizer, at least one preservative at least one vector or a mixture of these products for stimulating and increasing the immune response of said mammal.
• antigen denotes any natural or recombinant protein, in its native or mutated form, derived from a parasite or pathogenic agent other than Toxoplasma gondii capable of inducing in a mammal a cellular or humoral immune response.
• the purpose of combining the mutant strain of Toxoplasma gondii with such an antigen is to enhance the immune response of the mammal and thereby confer a better protection against an apicomplex infection.
• adjuvant any substance capable of enhancing and prolonging the immune response directed against the targeted antigen. The mechanism involved in making the immune response more effective is dependent on the adjuvant used.
• adjuvants are substances well known to those skilled in the art, among which include aluminum salts, squalene, saponins, constituents or bacterial toxins, or certain proteins (peptone, albumin, casein).
• stabilizer or preservatives denotes the compounds which make it possible to preserve the toxoplasma gondii strains in their packaging perfectly. The purpose of these compounds is to ensure the viability of T. gondii strains.
• Stabilizers or preservatives are substances that are well known to those skilled in the art, among which include carbohydrates (sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose) and polar organic solvents such as DMSO. (dimethylsulfoxide), polysorbates.
• vector is meant an entity used to penetrate a gene of interest into a cell. Vectors are substances well known to humans of Art, among which include nanoparticles, ISCOMs (ie “immune stimulatory complexes”), etc ...
• increasing the immune response is meant the activation of the different pathways of the mammalian immune system by the combination consisting of the mutant strain of T. gondii with a second antigen. It is to activate the innate immune response through an increase in the synthesis of cytokines of distinct categories such as interferons or interleukins. Interleukin-12 (IL-12) is able to stimulate Natural Killer (NK) cells. The cells thus stimulated will secrete interferon- ⁇ (IFN-gamma), which plays a major role in protecting against intracellular parasites such as T. gondii. In an adult mammal with a mature and competent immune system, the immune response is based on adaptive immunity (specific proliferation in response to foreign antigens by CD4 + and CD8 + T cells) in addition to the innate response.
• IFN-gamma Interferon- ⁇
• strains of Toxoplasma gondii isolated from their natural environment for their use according to the present invention may be associated with at least one antiparasitic compound or an antibiotic selected from the group comprising or consisting of pyrimethamine, sulfadiazine , cotrimoxazole, clindamycin, azithromycin or atovaquone.
• strains of Toxoplasma gondii isolated from their natural environment for their use according to the present invention may be associated with at least one antiparasitic compound or an antibiotic selected from the group comprising or consisting of pyrimethamine, the sulfadiazine, cotrimoxazole, clindamycin, azithromycin or atovaquone and, to a corticosteroid.
• an antibiotic selected from the group comprising or consisting of pyrimethamine, the sulfadiazine, cotrimoxazole, clindamycin, azithromycin or atovaquone and, to a corticosteroid.
• the present invention also relates to a method for preventing the onset and / or treating, in a mammal, ocular lesions caused by one or more apicomplexes of the family Sarcocystidae comprising a step of administering tachyzoites of one or more mutant strains of Toxoplasma gondii attenuated virulence to said mammal to reduce the number of ocular lesions.
• the mutant strains of Toxoplasma gondii have at least one adhesin MIC-1 or a MIC-3 adhesin inactivated by gene modification on at least one of the mic-1 or mic-3 genes.
• the mutant strains of Toxoplasma gondii have at least one MIC-1 adhesin and / or an inactivated MIC-3 adhesin by the deletion of at least one of the mic genes. - 1 and / or mic-3.
• the mutant strains of Toxoplasma gondii have both the MIC-1 and MIC-3 adhesins inactivated by a gene modification involving the two mic-1 and mic-3 genes. .
• the mutant strains of Toxoplasma gondii have both the MIC-1 and MIC-3 adhesins inactivated by the deletion of the two mic-1 and mic-3 genes.
• the mammal in the method according to the present invention, is a human being or an animal.
• the present invention also relates to a method for preventing the occurrence and / or treating, in a mammal, intraocular inflammation caused by one or more apicomplexes of the family Sarcocystidae comprising a step of administering tachyzoites of a strain or several mutant strains of Toxoplasma gondii of attenuated virulence to said mammal.
• the administration of tachyzoites of a strain or several mutant strains of Toxoplasma gondii is carried out enterally or parenterally.
• FIG 1A Ophthalmologic clinical signs in Swiss Webster (OF1) mice 4 weeks after challenge infection.
• mice The determination of ophthalmological clinical signs is performed under a binocular microscope on each eye of each of the mice 4 weeks after the mice have been orally infected with 50 cysts of the 76K strain of Toxoplasma gondii.
• the mice were vaccinated with 100 tachyzoites of the strain Toxo -3 KO honey 4 weeks before test infection (black column) or not (gray column).
• Figure 1B Ophthalmological clinical signs in Swiss Webster (OF1) mice 8 weeks after challenge infection.
• mice The determination of the ophthalmological clinical signs is performed under a binocular microscope on each eye of each of the mice 8 weeks after the mice have been infected orally with 50 cysts of the 76K strain of Toxoplasma gondii.
• the mice were vaccinated with 100 tachyzoites of the strain Toxo -3 KO honey 4 weeks before test infection (black column) or not (gray column).
• FIG. 1C Ophthalmologic clinical signs in Swiss Webster (OF1) mice 12 weeks after challenge infection.
• the mice were vaccinated with 100 tachyzoites of the strain Toxo honey -3 KO 4 weeks before test infection (black column) or not (gray column).
• FIG. 1A Evaluation of the number of intracerebral cysts in Swiss Webster (OF1) mice 4 weeks after challenge infection.
• Counting of the number of intracerebral cysts was performed from mouse brain morsel 4 weeks after the mice were orally infected with 50 cysts of Toxoplasma gondii strain 76K. Each point represents a mouse. Eight to ten counts are carried out on 10 of this crushed material using a binocular microscope on Malassez cells. The average is then calculated and reported to the entire initial volume to estimate the number of intracerebral cysts.
• Counting of the number of intracerebral cysts was performed from mouse brain mace 8 weeks after the mice were orally infected with 50 cysts of Toxoplasma gondii strain 76K. Each point represents a mouse. Eight to ten counts are carried out on 10 of this crushed material using a binocular magnifying microscope on Malassez cells. The average is then calculated and reported to the entire initial volume to evaluate the number of intracerebral cysts.
• FIG. 2C Evaluation of the number of intracerebral cysts in Swiss Webster (OF1) mice 12 weeks after challenge infection.
• Counting of the number of intracerebral cysts was performed from mouse brain mace 12 weeks after the mice were orally infected with 50 cysts of Toxoplasma gondii strain 76K. Each point represents a mouse. Eight to ten counts are carried out on 10 of this crushed material using a binocular magnifying microscope on Malassez cells. The average is then calculated and reported to the entire initial volume to estimate the number of intracerebral cysts.
• FIG. 3A Evaluation of the number of intra-retinal cysts in Swiss Webster (OF1) mice 4 weeks after challenge infection.
• Counting of the number of intra-retinal cysts was performed on the retinal-choroid complex of the enucleated eyes of the mice 4 weeks after the mice were orally infected with 50 cysts of the Toxoplasma gondii strain 76K. Each point represents a mouse.
• FIG. 3B Evaluation of the number of intra-retinal cysts in Swiss Webster (OF1) mice 8 weeks after challenge infection.
• Counting of the number of intra-retinal cysts was performed on the retinal-choroid complex of the enucleated eyes of the mice 8 weeks after the mice were orally infected with 50 cysts of the Toxoplasma gondii strain 76K. Each point represents a mouse.
• FIG. 3C Evaluation of the number of intra-retinal cysts in Swiss Webster (OF1) mice 12 weeks after challenge infection.
• Counting of the number of intra retinal cysts was performed on the retinal-choroid complex of the enucleated eyes of the mice 12 weeks after the mice were orally infected with 50 cysts of the Toxoplasma gondii strain 76K. Each point represents a mouse.
• Figure 4A assay of PIFN- ⁇ in the Swiss Webster (OF1) mouse aqueous humor 4 weeks after the start of the challenge infection.
• the assay of gamma interferon (IFN-gamma) in the aqueous humor was performed 4 weeks after the mice were orally infected with 50 cysts of the 76K strain of Toxoplasma gondii. Each point represents a mouse,
• Figure 4B IFN-gamma assay in the Swiss Webster (OF1) mouse aqueous humor 8 weeks after the start of the challenge infection.
• the interferon gamma (IFN-gamma) assay in the aqueous humor was performed
• mice 8 weeks after mice were orally infected with 50 cysts of Toxoplasma gondii strain 76K. Each point represents a mouse
• Figure 4C Assay of IFN- ⁇ in Swiss Webster (OF1) mouse aqueous humor 12 weeks after the start of challenge infection.
• the interferon-gamma (IFN-gamma) assay in the aqueous humor was performed 12 weeks after the mice were orally infected with 50 cysts of the Toxoplasma gondii strain 76K. Each point represents a mouse.
• Figure 5A Ophthalmological clinical signs in Swiss Webster (OF1) mice 4 weeks old and infected in utero with Toxoplasma gondii.
• Stage 2 Tyndall in anterior or severe vitreous chamber and / or dilation of the iris and / or conjunctivo-scleral vessels,
• Each point represents the cumulative clinical stage for a young mouse (cumulative clinical stage being the sum of the clinical stages of the two eyes per mouse).
• mice lots of Swiss Webster (OF1) mice:
• Figure 5B Ophthalmological clinical signs in Swiss Webster (OF1) mice, 8 weeks old, infected in utero with Toxoplasma gondii.
• Stage 2 Tyndall in anterior or severe vitreous chamber and / or dilation of the iris and / or conjunctivo-scleral vessels,
• Each point represents the cumulative clinical stage for a young mouse (cumulative clinical stage being the sum of the clinical stages of the two eyes per mouse).
• mice lots of Swiss Webster (OF1) mice:
• Figure 6 Evaluation of the number of intracerebral cysts in Swiss Webster (OF1) mice, 8 weeks old, infected in utero with Toxoplasma gondii.
• Counting of the number of intracerebral cysts was made from a brain seed of 8-week-old suckling mice whose mothers were or were not infected at 12 gestation with 15 cysts of Toxoplasma gondii strain 76K. Eight to ten counts are made on 10 of this crushed material using a binocular loupe on Malassez cells. The average is then calculated and reported to the entire initial volume to evaluate the number of intracerebral cysts. Each point represents a mouse.
• Figure 7 Evaluation of the number of intra-retinal cysts in Swiss Webster (OF1) mice, 8 weeks old, infected in utero with Toxoplasma gondii.
• Example 1 Efficacy of the strain Toxo micl-3 KO in the prevention of toxoplasmosis in a mouse model of chronic ocular toxoplasmosis
• Vaccination is performed on Swiss Webster (OF1), non-consanguine, female, 8 week old mice from the January breeding center (Le Genest-Saint-Isle, France). The mice are maintained throughout the confinement level 2 animal facility experiment in order to minimize the risk of external contamination.
• OF1 Swiss Webster
• non-consanguine female
• 8 week old mice from the January breeding center (Le Genest-Saint-Isle, France).
• the mice are maintained throughout the confinement level 2 animal facility experiment in order to minimize the risk of external contamination.
• the mutant strain of Toxoplasma gondii, Toxo micl-3 KO, whose genes coding for the MIC1 and MIC3 proteins were invalidated, is maintained by successive passages on a human foreskin fibroblast line (HFF) grown in DMEM medium (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 U / mL streptomycin.
• HFF human foreskin fibroblast line
• the tachyzoites are recovered in the supernatant and counted on Malassez cell. The concentration is then adjusted to obtain a final dose of 100 tachyzoites in 200 DMEM medium, corresponding to the mouse vaccine dose.
• the wild strain RH of Toxoplasma gondii is also maintained by successive passages on a line of human foreskin fibroblasts (HFF) cultured in DMEM medium to which fetal calf serum is added. %, 2 mM L-glutamine, 100 U / mL penicillin and 100 U / mL streptomycin.
• HFF human foreskin fibroblasts
• the tachyzoites of strain RH are washed, sonicated twice at 60 watt / s for 10 min in ice and centrifuged at 2000 g for 30 min at + 4 ° C. The supernatant is recovered and the concentration is determined by an assay kit (BCA assay) that uses as standard Bovine Albumin Serum (BSA). The aliquots are stored at -20 ° C.
• BCA assay Bovine Albumin Serum
• the Type II 76K strain is used for the mouse challenge infection. This strain is conserved as cysts by continuous passages on CBA / J mice by gavage (mouse cycle). On the eve of the challenge infection, a cycle mouse, infected at least two months before with 76K strain cysts, is sacrificed by cervical dislocation.
• mice The brains of mice are recovered after washing with alcohol from the coat of the animal's head. A cutaneous incision with scissors is performed at the level of a retro-auricular nuchal line. The scalp is then anteverted and two craniotomies beginning at the occipital foramen to the frontal bones are performed. The cranial vault is removed and the brain is removed in its entirety. The brain is then ground in 5 mL of RPMI medium in a potter. The ground material is left overnight at + 4 ° C. After counting Malassez cell cysts, the concentration is adjusted to obtain infection doses of 200 each containing 50 cysts.
• mice from lots (i) and (ii) were subjected to gavage challenge infection using an 18 gauge cannula with 50 cysts of Toxoplasma gondii strain 76K. , prepared in accordance with the description in paragraph 1.2.3.
• the serological status of the mice is determined by an indirect type ELISA test.
• the blood samples are centrifuged at 5,000 g for 15 min and the serum is recovered.
• the total parasitic extract of the RH strain whose preparation is described in section 1.2.2 is diluted in a pH9.6 carbonate buffer to obtain a final concentration of 10 ⁇ g / mL.
• Flat-bottomed 96-well plates are then sensitized overnight at + 4 ° C. by depositing in each well 100 of total Toxoplasma gondii extract.
• the plates are then washed three times with the washing buffer (PBS 1x - Tween 20 0.05%) and saturated for 1 h 30 at 37 ° C with a solution of PBS IX - Tween 20 0.05% supplemented with 4% Serum Bovine Albumin (BSA) (Sigma). The medium is then removed.
• PBS 1x - Tween 20 0.05%
• BSA Serum Bovine Albumin
• the sera to be tested are diluted to l / 50th in a solution of PBS IX - 0.05% Tween 20) and are deposited in duplicate in the wells. After one hour of incubation at 37 ° C. and a new washing series, the secondary anti-mouse IgG antibody coupled to the alkaline phosphatase (Sigma A3562, goat anti-Mouse IgG) and diluted to 1/5000 eme is deposited at the rate of 100 per well. The samples are then incubated for one hour at 37 ° C.
• ophthalmological analyzes are carried out using a binocular microscope (Zeiss OPMI 99 colposcope foot on which is fixed a binocular head Zeiss F 170 with its two objectives) after instillation of two drops of mydriactum 0, 1% in each eye at 10 min intervals.
• the examination of the fundus is then performed using the same tool with the help of a lens "superfluid" of 90 diopters.
• the animal is brought under the light beam with a zoom of 0.6.
• the eye is centered, then the lens is brought 5 mm from the eyeball without coming into contact with it.
• the plate After washing with 0.05% PBS-Tween 20 buffer, the plate is saturated for 1h at room temperature with saturation buffer (IX PBS - 10%> FCS) at 200 per well. After a further series of three washes, the aqueous humor samples diluted to l / 10th in saturation buffer are deposited and subsequently incubated for two hours at room temperature at 50 per well. In parallel, a range is made from murine IFN- ⁇ obtained commercially. After a further series of wash, 50 of the solution containing the antibody and the detection of enzyme are deposited at a dilution of l / 250 th in the saturation buffer and incubated for lh.
• saturation buffer IX PBS - 10%> FCS
• the plate is revealed with the substrate (Tetramethylbenzidine, Sigma), at a rate of 50 ⁇ l per well. After 30 min, 25 ⁇ l of stop solution (H 2 S0 4 2N) are added. The optical densities are read at 450 nm using a plate reader (Multiskan MCC340 Wallace).
• mice are recovered after washing with alcohol from the coat of the animal's head. A cutaneous incision with scissors is performed at the level of a retro-auricular nuchal line. The scalp is then anteverted and two craniotomies beginning at the occipital foramen to the frontal bones are performed. The cranial vault is removed and the brain is removed in its entirety. The brain is then ground in 5 mL of RPMI medium in a potter. The ground material is left overnight at + 4 ° C.
• the eyes of the mice are enucleated. Under a binocular loupe, complete conjunctival debridement is practiced. A limbal incision is made and the cornea is extracted. Four scleral orthogonal incisions are then made to the posterior pole of the Vannas scissors and the retina-choroid-scleral assembly is spread flat on the work surface. The lens is also removed by maximizing the vitreous gel in contact with the retina to minimize trauma. The retina-choroid complex is then gently removed using a micromanipulator and placed in 50 RPMI medium, then homogenized by "back and forth" in the cone of a micropipette.
• mice Thirty days after vaccination, the mice were challenged by gavage with 50 cysts of the 76K strain.
• mice in the control group remain seronegative.
• a second serological test was performed one month after the challenge infection. After the challenge infection, all mice in the vaccinated and infected lot (i) and the infected lot (ii) are seropositive. On the other hand, the optical density observed from the sera of the vaccinated and infected mice is higher than that observed from the mice only vaccinated or uninfected, thus reflecting a higher antibody level in the vaccinated and infected mice compared to that observed. in mice only vaccinated or uninfected.
• Vaccination is performed on Swiss Webster (OF1), non-consanguine, female, 8 week old mice from the January breeding center (Le Genest-Saint-Isle, France). The mice are maintained throughout the confinement level 2 animal facility experiment in order to minimize the risk of external contamination.
• the mutant strain of Toxoplasma gondii, Toxo micl-3 KO, whose genes coding for the MIC1 and MIC3 proteins were invalidated, is maintained by successive passages on a line of human foreskin fibroblasts (HFF) grown in DMEM medium (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 U / mL streptomycin.
• HFF human foreskin fibroblasts
• the tachyzoites are recovered in the supernatant and counted on Malassez cell. The concentration is then adjusted to obtain a final dose of 100 tachyzoites in 200 DMEM medium, corresponding to the mouse vaccine dose.
• the wild strain RH of Toxoplasma gondii is also maintained by successive passages on a line of Human foreskin fibroblasts (HFF) grown in DMEM medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 U / mL streptomycin.
• HFF Human foreskin fibroblasts
• the tachyzoites of strain RH are washed, sonicated twice at 60 watts / sec for 10 min in ice and centrifuged at 2,000 g for 30 min at + 4 ° C. The supernatant is recovered and the concentration is determined by a kit of assay (BCA assay) which uses as standard Bovine Serum Albumin (BSA). The aliquots are stored at -20 ° C.
• BCA assay Bovine Serum Albumin
• the Type II 76K strain is used for the mouse challenge infection. This strain is conserved as cysts by continuous passages on CBA / J mice by gavage (mouse cycle). On the eve of the challenge infection, a cycle mouse, infected two months earlier by 76K strain cysts, is sacrificed by cervical dislocation.
• mice The brains of mice are recovered after washing with alcohol from the coat of the animal's head. A cutaneous incision with scissors is performed at the level of a retro-auricular nuchal line. The scalp is then anteverted and two craniotomies beginning at the occipital foramen to the frontal bones are performed. The cranial vault is removed and the brain is removed in its entirety. The brain is then ground in 5 mL of RPMI medium in a potter. The ground material is left overnight at + 4 ° C.
• the cysts are then counted on Malassez cell and the concentration is adjusted to obtain infection doses of 200 each containing 15 cysts.
• mice Female Swiss-OF1 mice are divided into three distinct lots:
• lot (iii) consisting of 15 unvaccinated and infected female mice (unvaccinated / infected lot). Twenty-eight days after vaccination (D28), submaxillary blood sampling was performed on all vaccinated mice to diagnose seroconverted mice (vaccinated / infected lot - lot (i)).
• mice diagnosed with the vaccinated / infected lot (i) and the unvaccinated / infected lot (lot iii) were challenged by gavage an 18 gauge cannula with 15 cysts of strain 76K, the preparation of which is described in section 1.2.3.
• mice Four weeks after farrowing, the young mice benefit from an ophthalmological examination performed under general anesthesia obtained by inhalation of 2.5% isoflurane gas (oxygen at 3 l per minute) in an induction cage (Mark 5, Minerve SA) for 2 to 5 minutes, in groups of up to 5 mice. Eight weeks after farrowing, young mice are sacrificed and a second ophthalmological examination is performed. Ocular and cerebral cysts are counted and the intraocular immune response is analyzed.
• isoflurane gas oxygen at 3 l per minute
• the serological status of the mice is determined by an indirect type ELISA test. For mothers, serological status was studied 4 weeks after vaccination and 4 and 8 weeks after challenge infection. For young mice, serological status was studied in the fourth and eighth week of life.
• the blood samples are centrifuged at 5,000 g for 15 min and the serum is recovered.
• the total extract of the RH strain whose preparation is described in section 1.2.2 is diluted in a carbonate buffer pH9.6 to obtain a final concentration of 10 ⁇ g / mL.
• 96-well flat-bottomed plates are then sensitized overnight at + 4 ° C by a deposit in each well of 100 ⁇ , total extract of Toxoplasma gondii.
• the plates are then washed three times with the washing buffer (PBS 1 ⁇ - Tween 20 0.05%) and then saturated for 1 h 30 min at 37 ° C. with a solution of PBS IX-Tween 20 0.05% supplemented with 4% of serum.
• Bovine albumin (BSA) (Sigma). The medium is then removed.
• the sera to be tested are diluted to l / 50th in a solution of PBS IX - 0.05% Tween 20) and are deposited in duplicate in the wells. After one hour of incubation at 37 ° C. and a new washing series, the secondary anti-mouse IgG antibody coupled to the alkaline phosphatase (Sigma A3562, goat anti-Mouse IgG) and diluted to 1/5000 eme is deposited at the rate of 100 per well. The samples are then incubated for one hour at 37 ° C.
• ophthalmological analyzes are performed using a binocular loupe (Zeiss OPMI 99 colposcope foot on which is fixed a binocular head Zeiss F 170 with its two objectives) after instillation of two drops of Mydriactum ® 0.1% in each eye.
• a binocular loupe Zeiss OPMI 99 colposcope foot on which is fixed a binocular head Zeiss F 170 with its two objectives
• Anterior segment examination is performed with respect to corneal hydration to avoid the occurrence of exposure keratitis or corneal edema that may create artifacts.
• Ophthalmological examination revealed the occurrence of anterior uveitis (pigmented retro-corneal precipitates) and / or cataracts (crystalline opalescence, sutural or subcapsular cataracts or even a true nuclear cataract), hyalitis or retinal hemorrhages. In order to evaluate inflammation, the following rating was used (Sauer et al., 2009 Journal of Ophthalmology, 32: 742-749):
• mice The brains of mice are recovered after washing with alcohol from the coat of the animal's head. A cutaneous incision with scissors is performed at the level of a retro-auricular nuchal line. The scalp is then anteverted and two craniotomies beginning at the occipital foramen to the frontal bones are performed. The cranial vault is removed and the brain is removed in its entirety. The brain is then ground in 5 mL of RPMI medium in a potter. The ground material is left overnight at + 4 ° C.
• mice After taking aqueous humor, the mice are enucleated. Under a binocular loupe, complete conjunctival debridement is practiced. A limbal incision is made and the cornea is removed. Four scleral orthogonal incisions are then made to the posterior pole of the Vannas scissors and the retina-choroid-scleral set is spread flat on the work surface. The lens is also removed leaving the vitreous gel in maximum contact with the retina to minimize trauma. The retina-choroid complex is then gently removed using a micromanipulator and placed in 50 of RPMI medium, then homogenized by "going back and forth" in the cone of a micropipette. For the counting of cysts, the entire sample is observed using a binocular loupe between blade and coverslip.
• an aqueous humor sample is taken under a binocular loupe using a 30 gauge needle mounted on a 1 ml syringe.
• the two samples corresponding to the two eyes of the same mouse are combined and intra-ocular IFN- ⁇ is assayed by ELISA (BD Opt EIA Mouse IFN- ⁇ ELISA set).
• ELISA BD Opt EIA Mouse IFN- ⁇ ELISA set.
• a J- 1 the capture antibody diluted to l / 250th in buffer "coating" (dilution buffer: 85 mM NaHC0 3, 15 mM Na 2 C0 3, pH 9.5) is deposited on plate 96 wells. These plates are incubated at + 4 ° C all night.
• the plate After washing with 0.05% PBS-Tween 20 buffer, the plate is saturated for 1h at room temperature with saturation buffer (1X PBS, 10% FCS) at 200 per well. After a further series of three washes, the aqueous humor samples diluted to l / 10th in saturation buffer are deposited and subsequently incubated for two hours at room temperature (50 per well to IFN- ⁇ ). In parallel, a range is made from murine IFN- ⁇ obtained commercially. After a further series of wash, 50 of the solution containing the antibody and the detection of enzyme are deposited at a dilution of l / 250 th in the saturation buffer and incubated for lh.
• saturation buffer (1X PBS, 10% FCS)
• the plate is revealed with the substrate (tetramethylbenzidine), at 50 ⁇ , per well. After 30 minutes, 25 ⁇ l of stop solution (H 2 SC "4 2N) are added, and the optical densities are read at 450 nm using a plate reader (Multiskan MCC340 Wallace).
• mice were vaccinated, put to males and infected at mid-pregnancy with a wild-type strain.
• T. gondii The mice of lot (i), as well as the control mice of lots (ii) and (iii) were put to males. Then, mice from lots (i) and (iii) were subjected to challenge infection orally at twelfth day of gestation with 15 cysts of T. gondii strain 76K.
• Perinatal mortality was assessed during the first 4 weeks of life. Thus, during this period, 6 out of 127 mice died in the lot (i) vaccinated, ie 4.72%.
• a brain sample was taken from 121 suckling mice in lot (i), 36 mice from batch (ii) (6 mice not removed) and 50 mice from lot (iii) (6 not taken). A significant difference is observed between the number of mice with cerebral infection in lot (i) (36.4%>) and the number of mice with cerebral infection in lot (iii) (98%>) (p ⁇ 0.0001). No cerebral cysts were detected in the young mice (ii).
• a young mice is considered infected at the eye level when at least one cyst is observed retinal in at least one of the eyes.
• the rate of ocular infection is very significantly less in the young mice of the lot (i) (24% o the mice infected with the ocular level) than in the mice of the lot (iii) (71.4%) of the infected mice at the eye level. ). None of the mice in lot (ii) had an intra-retinal cyst.
• the number of mean intraspinal cysts per mouse ( Figure 7) is also very significantly lower in the animals in batch (i) (0.56 ⁇ 1.18 cysts per eye) than in the animals in the batch ( iii) (2.54 ⁇ 4.10 cysts per eye).
• the mean IFN- ⁇ concentration in the anterior chamber was 1,170.5 ⁇ 1,918.7 pg / mL in the mice of lot (i), of 2,147.6 ⁇ 3,917.5 pg / mL in the mice of the batch (iii ) and 940.9 ⁇ 180.0 pg / mL in the young mice of lot (ii).

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