Classifications

• • 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
  • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
• • 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
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • 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
  • A61P33/06—Antimalarials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
  • C07K14/445—Plasmodium
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to the field of medicine, and more particularly that of the fight against malaria. Technological background of the invention
• Malaria is an infectious disease caused by a eukaryotic unicellular parasite of the genus Plasmodium. This parasitic disease is worldwide and causes serious economic and health problems in developing countries.
• P. falciparum is the most deleterious species of the five types of Plasmodium that infect humans. According to the World Health Organization (WHO), P. falciparum is responsible for 250 to 500 million cases of acute illness and about 1 million deaths each year (mostly children under 5 and pregnant women). ).
• the cerebral malaria is a severe neurological complication of malaria responsible for the vast majority of fatal cases of the disease. Even if the individual survives, cerebral malaria can lead to severe neurological sequelae, especially in young children whose immune systems are being formed.
• cerebral pathology is probably the result of the sequestration of parasitized red blood cells in the micro-vessels of the main organs (spleen, lungs, heart, intestines, kidneys, liver and brain) and the production of pro-inflammatory cytokines in these same organs, leading to a systemic syndrome and condition that can lead to the death of the individual.
• GAP Genetically Attenuated Parasites
• PNP purine nucleoside phosphorylase
• NT1 nucleoside transporter 1
• the objective of the present invention is to provide novel vaccine compositions for preventing malaria, and more particularly the appearance of severe malaria infections such as cerebral malaria.
• the inventors have demonstrated that a live parasite belonging to the genus Plasmodium, and in which the function of the hmgb2 gene has been inactivated, could be used as an immunogen in a malaria vaccine. They have observed that the administration of such a parasite induces an immune reaction in the host which makes it possible to obtain both the clearance of the parasite (parasite load below the threshold of detection by microscopy in the peripheral blood ) but also an effective and long-term protection against infection by Plasmodium, particularly by a highly pathogenic strain, and more particularly against the erythrocyte phase of the parasite which is responsible for the symptomatology of the disease and its transmission.
• the present invention relates first of all to a living parasite belonging to the genus Plasmodium in which the function of the hmgb2 gene is inactivated for use in the prevention of malaria, and more particularly in the prevention of neuropaludism which is a severe neurological complication of disease.
• the parasite according to the invention is used in the prevention of malaria or neuropaludism in a mammal, in particular a human.
• the strain of the parasite may be selected from the group consisting of Plasmodium berghei, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
• the strain of the parasite is selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi, more particularly preferably in the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. .
• the strain of the parasite is Plasmodium falciparum.
• the strain of the parasite is Plasmodium berghei, in particular Plasmodium berghei NK65.
• the parasite in its wild form does not cause neuropaludism.
• the strain of the parasite is a strain of
• the parasite according to the invention may be in intra-erythrocyte form, in particular in the form of trophozoites, merozoites or intra- erythrocytic schizonts, preferably in the form of merozoites or intra- erythrocytic schizonts.
• the parasite according to the invention may be in the form of non-intra-erythrocyte merozoites, that is to say merozoites obtained from parasitized red blood cells by total or partial purification.
• the parasite according to the invention may also be in the form of sporozoites.
• the function of the hmgb2 gene can be inactivated by total or partial deletion of said gene, preferably by total deletion of said gene.
• the coding region of the hmgb2 gene may be replaced by a selection marker, such as the human gene coding for dihydrofolate reductase.
• the function of the hmgb2 gene can also be inactivated by means of an interfering AR blocking or decreasing the translation of the HMGB2 protein.
• the function of one or more other genes can be inactivated.
• the other gene or genes whose function is inactivated can be chosen from the group consisting of the genes encoding the purine nucleoside phosphorylase, the nucleoside transporter 1, UIS3, UIS4, p52 and p36, and a combination thereof. .
• the present invention relates to an immunogenic composition
• an immunogenic composition comprising, as an immunogen, an immuno logically effective amount of a parasite according to the invention, and one or more pharmaceutically acceptable excipients or carriers.
• the present invention also relates to a vaccine against malaria comprising, as an immunogen, a parasite according to the invention, and one or more pharmaceutically acceptable excipients or carriers.
• the immunogenic composition or the vaccine may also comprise one or more immunological adjuvants.
• the immunological adjuvant (s) may be selected from the group consisting of muramyl peptide adjuvants; trehalose dimycolate (TDM); lipopolysaccharide (LPS); monophosphoryl lipid A (MPL); carboxymethylcellulose; the complete adjuvant of Freund; incomplete Freund's adjuvant; adjuvants of "oil-in-water” emulsion type optionally added with squalene or squalane; mineral adjuvants; bacterial toxins; CpG oligodeoxynucleotides; saponins; synthetic copolymers; cytokines and imidazoquinolones.
• TDM trehalose dimycolate
• LPS lipopolysaccharide
• MPL monophosphoryl lipid A
• carboxymethylcellulose the complete adjuvant of Freund; incomplete Freund's adjuvant; adjuvants of "oil-in-water” emulsion type optionally added with squalene or s
• the immunogenic composition or vaccine may be formulated for parenteral administration.
• the immunogenic composition or the vaccine may comprise several parasites according to the invention.
• the composition or the vaccine comprises between 10 and 10 7 , preferably between 10 and 10 5 , and more preferably between 10 2 and 10 5 parasites per injection dose.
• the immunogenic composition or the vaccine comprises between 10 and 10 6 , preferably between 10 2 and 10 6 , parasites according to the invention in an intra-erythrocyte form per unit of dose.
• the immunogenic composition or the vaccine comprises between 10 2 and 10 7 parasites according to the invention in the form of non-intra-erythrocyte merozoites per unit of dose.
• the immunogenic composition or the vaccine comprises between 10 3 and 10 7 parasites according to the invention in the form of sporozoites per unit of dose.
• the present invention relates to the use of a parasite according to the invention, for the preparation of a vaccine composition against malaria or cerebral malaria.
• the present invention also relates to a method for immunizing a subject against malaria, comprising administering an immunogenic composition or vaccine according to the invention to said subject, preferably parenterally, particularly by subcutaneously, intramuscularly, intradermally or intravenously.
• Figure 1 Parasitaemia of C57BL / 6 mice infected with parasitized red blood cells (GRP) with the wild-type parasite i3 ⁇ 4NK65 or the mutated parasite 3 ⁇ 4NK65 Ahmgb2 (10 5 and 10 6 GRP by intravenous injection, respectively). Parasitaemia is analyzed by microscopic counting of GRP in stained smears and presented as the percentage of GRP (mean ⁇ standard deviation). The experiment was done with 5 mice and repeated several times. The p value is 0.008.
• GRP parasitized red blood cells
• Figure 2 Study of the survival of the first-infected mice with the mutated parasite i3 ⁇ 4NK65 Ahmgb2 and then infected three times with the two wild pathogenic parasites i3 ⁇ 4NK65 and i3 ⁇ 4ANKA. The first test was done at day 19 (19 days after primary infection), the second at day 71 and finally the third day at day 166. At each challenge, the pathogenicity of the two GRP preparations infected with i3 ⁇ 4NK65 and i3 ⁇ 4ANKA was verified by infecting non-primo-infected mice of the same age and following their fate and death by hyperparasitaemia or neuropaludism. Five mice were analyzed for each experiment.
• FIG. 3 Immunization of C57BL / 6 mice with different amounts of GRP infected with the mutated parasite 3 ⁇ 4NK65 Ahmgb2.
• Ten mice were infected at time 0 with 10 3 ( ⁇ ), 10 4 (o) or 10 5 ( ⁇ ) GRP infected with i3 ⁇ 4NK65 Ahmgb2.
• Na ⁇ ve mice were also challenged on day 24 with 10 5 GRPs infected with the wild-type pathogen i3 ⁇ 4NK65 (A) or i3 ⁇ 4ANKA ( ⁇ ).
• HMGB1 protein In situations of danger for the cell (foreign body, inflammation), these proteins are secreted actively in the cellular medium and / or passively in case of cell necrosis, and act as real danger signals (alarmins) by activating macrophages and other competent cells of the immune system (Lotze and Tracey, 2005).
• alarmins real danger signals
• the involvement of human HMGB1 protein has been demonstrated in diseases causing systemic inflammation such as lupus erythematosus, septic shock or rheumatoid arthritis, and in some cancers.
• the increase in human HMGB1 protein in patients' sera has been correlated with the severity of these diseases.
• PfHMGB1 and PfHMGB2 small proteins of less than 100 amino acids. These two proteins, like their human counterparts, are capable of bending DNA and interacting with particular DNA structures (Briquet et al., 2006). They are also secreted and found in culture supernatants of P. falciparum.
• the HMGB1 and 2 proteins of P. falciparum are capable of activating and inducing TNFa expression in mouse monocyte lines (Kumar et al., 2008).
• the inventors have used C57BL / 6 mice infected with the P. berghei ANKA (i3 ⁇ 4ANKA) parasite as an animal model of neuropaludism.
• This animal model reproduces the main characteristics of human pathology such as ataxia, disorientation in space, convulsions and coma. These clinical symptoms can lead to the death of the animal.
• we observe in this model the sequestration of parasitized red blood cells, haemorrhages and brain lesions, the production of pro-inflammatory cytokines and the destruction of the blood-brain barrier.
• all C57BL / 6 mice infected with the i3 ⁇ 4ANKA isolate die within about 7 days of cerebral malaria.
• the inventors have observed that 60% of the mice infected with the ANKA Ahmgb2 parasite do not develop neuropaludism.
• the inventors inactivated the hmgb2 gene of another P. berghei isolate, the NK65 isolate, which induces the death of C57BL / 6 mice by hyper-parasitaemia within 20 to 25 days after infection but does not cause cerebral malaria. They have first observed that the inactivation of this gene hampers the development of i3 ⁇ 4NK65 Ahmgb2 in the mouse with a clearance of the parasite in the peripheral blood obtained 10 to 15 days after infection.
• the inventors have then demonstrated that the host response leading to the clearance of the parasite was sufficient to induce sterile immunity of more than six months, not only vis-à-vis the homologous wild strain 3 ⁇ 4NK65 but also vis-a-vis vis-à-vis the highly pathogenic heterologous i3 ⁇ 4ANKA strain that is capable of inducing cerebral malaria.
• the present invention relates to a living parasite belonging to the genus Plasmodium in which the function of the hmgb2 gene is inactivated for use in the prevention of malaria or cerebral malaria, preferably in a mammal, and more particularly favorite in a human.
• the parasite is preferably capable of developing in vertebrates, and more particularly in mammals, and belongs to the subgenus selected from the group consisting of Plasmodium vinckeia, Plasmodium plasmodium and Plasmodium laverania.
• the parasite is capable of developing in a human host and belongs to the subgenus Plasmodium plasmodium or Plasmodium laverania.
• the parasite belongs to a species responsible for malaria in humans, more particularly to a species selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
• Plasmodium knowlesi is a parasite that mainly infects primates, more and more cases of human infections with this parasite are reported.
• the parasite belongs to a species selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. According to a particular embodiment, the parasite belongs to a species selected from the group consisting of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. According to a preferred embodiment, the parasite belongs to the species Plasmodium falciparum.
• the parasite belongs to a species that is capable of inducing an immune reaction but is not capable of provoking symptoms of malaria in humans.
• this parasite is a parasite of rodents belonging to the subgenus Plasmodium vinckeia.
• the use of rodent parasites in the context of vaccination in humans considerably reduces the risks associated with the administration of live parasites in the subject.
• the rodent parasite may be modified to express one or more proteins of a human-infective Plasmodium, such as P. falciparum, which is or is necessary for the invasion of human red blood cells. Such proteins are for example described in the article by Triglia et al., 2000.
• the parasite belongs to the species Plasmodium berghei or Plasmodium yoelii. More preferably, the parasite belongs to the species Plasmodium berghei. According to a preferred embodiment, the parasite is the NK65 isolate of the Plasmodium berghei species.
• the parasite belongs to a species selected from the group consisting of Plasmodium berghei, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
• the parasite may also belong to a species selected from the group consisting of Plasmodium berghei, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae or in the group consisting of Plasmodium berghei, Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae, or in the group group consisting of Plasmodium berghei and Plasmodium falciparum.
• the wild strain of the parasite does not cause neuropaludism.
• This strain may be, for example, chosen from the group consisting of Plasmodium berghei NK65, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
• This strain may also be a strain of Plasmodium falciparum having lost its cyto-adhesion capacity or having a reduced cyto-adhesion capacity.
• the wild strain of the parasite is a non-cyto-adherent Plasmodium falciparum strain.
• the wild strain of the parasite is a strain of Plasmodium falciparum having a reduced cyto-adhesion capacity.
• Cyto-adhesion is a property of Plasmodium falciparum that is directly related to the development of neuropaludism.
• red blood cells infected with cytoplasmic Plasmodium falciparum strain have the ability to bind to endothelial cell surface molecules such as CD36, ICAM1, VCAM1 or PECAM1 / CD31, and thus to induce vascular obstruction, inflammation and damage to different organs, especially at the brain level.
• the cyto-adhesion capacity of a strain can be evaluated by any technique known to those skilled in the art, such as, for example, that described in the article by Buffet et al, 1999 or that of Traoré et al. 5, 2000.
• the term "reduced cyto-adhesion capacity" refers to a lower cyto-adhesion capacity than that observed on a cytoplasmic Plasmodium reference strain, for example the Plasmodium falciparum strain 3D7.
• the cyto-adhesion can be reduced by at least 40, 50, 60, 70, 80, 90 or 95%, preferably at least 80%, and more preferably at least 80%.
• Plasmodium falciparum strain 3D7 a cytoplasmic Plasmodium reference strain
• the wild strain of the parasite is a strain of Plasmodium falciparum little or no cyto-adherent.
• the wild strain of the parasite may be a strain of Plasmodium falciparum with reduced cyto-adhesion ability selected from the group consisting of Plasmodium falciparum strain in which the clagC gene is inactivated, Plasmodium falciparum D10 strain and Plasmodium falciparum strain T9-96.
• the function of the hmgb2 gene is inactivated.
• This inhibition can be obtained by many methods well known to those skilled in the art. It is thus possible to block the function of the gene at the transcriptional, translational or protein level, for example by blocking or reducing the transcription or translation of the hmgb2 gene or by disrupting the correct folding of the gene.
• the protein or its activity is the protein or its activity.
• the function of the hmgb2 gene can in particular be inactivated by the total or partial deletion of this gene, or the insertion or substitution of one or more nucleotides so as to render this gene inactive.
• the function of the hmgb2 gene is inactivated by total or partial deletion of this gene, preferably by total deletion.
• the deletion of the hmgb2 gene is obtained by homologous recombination.
• This method is well known to those skilled in the art and has been applied many times to parasites of the genus Plasmodium (see for example Thathy and Ménard, 2002).
• the coding region of the hmgb2 gene is replaced by homologous recombination by a marker making it possible to select the parasites in which the recombination has taken place.
• the selection marker may be, for example, the human dihydrofolate reductase (dhfr) gene which confers resistance to pyrimethamine on the parasite.
• the parasite used is a parasite in which the hmgb2 gene has been replaced by a selection marker, preferably by the human dhfr gene.
• the parasite is a Plasmodium berghei, preferably the NK65 isolate, in which the hmgb2 gene has been replaced by a selection marker, preferably by the human dhfr gene.
• the parasite used is a Plasmodium falciparum, preferably not or slightly cyto-adherent, in which the hmgb2 gene has been replaced by a selection marker, preferably by the human gene dhfr.
• the function of the hmgb2 gene can also be inactivated by blocking or decreasing the translation of the mRNA of this gene.
• the interference with RNA which makes it possible to specifically inhibit the expression of the target gene, is a phenomenon well known to those skilled in the art which has already been used to inhibit the expression of Plasmodium genes (see for example, McRobert and McConkey, 2002, Mohmmed et al., 2003 and Gissot et al., 2004).
• a sequence coding for an interfering RNA, or its precursor is introduced into the genome of the parasite and its expression is controlled by a strong, preferably constitutive, promoter, such as, for example, the promoter of the d eEF1cc elongation that is active in all stages of parasite development, or that of the HSP70 gene that is active in sporozoites and during the erythrocyte cycle.
• a strong, preferably constitutive, promoter such as, for example, the promoter of the d eEF1cc elongation that is active in all stages of parasite development, or that of the HSP70 gene that is active in sporozoites and during the erythrocyte cycle.
• the sequence and structure of the interfering RNA can be readily selected by those skilled in the art.
• the interfering RNA used may be a small interfering RNA (siRNA).
• hmgb2 genes of Plasmodium species not yet sequenced are easily identifiable by methods well known to those skilled in the art, in particular by hybridization or PCR.
• the function of one or more genes, other than hmgb2 can also be inactivated.
• the additional gene whose function is inactivated may be a gene involved in parasite survival in a mammalian host, particularly in humans.
• the inactivation of this additional gene makes it possible to attenuate the virulence of the parasite while preserving its immunogenic nature.
• This additional gene may be selected from the group consisting of purine nucleoside phosphorylase (PNP; PFE0660c), nucleoside transporter 1 (NT1; PF13 0252), UIS3 (PF130012), UIS4 (early transcript PF10 0164), p52 (PFD0215c 6-cysteine motif protein) and p36 (PFD0210c), as well as combinations thereof (references in parenthesis are the PlasmodB library entry numbers of Plasmodium falciparum 3D7 sequences, given here as an example ).
• PNP purine nucleoside phosphorylase
• NT1 nucleoside transporter 1
• UIS3 PF130012
• UIS4 early transcript PF10 0164
• p52 PD0215c 6-cysteine motif protein
• p36 p36
• the parasites according to the invention are used in erythrocyte form, more particularly in the form of non-intraerythrocytic merozoites or in the form of merozoites, trophozoites or intra- erythrocytic schizonts.
• the parasites according to the invention are used in the form of merozoites, trophozoites or intra- erythrocytic schizonts, that is to say within red blood cells.
• the parasites are used in the form of non-intra-erythrocyte merozoites, that is to say merozoites partially or totally purified after rupture of parasitized red blood cells.
• Merozoites can be obtained according to any of the methods known to those skilled in the art such as that described in the article by Boyle et al., 2010.
• the parasitized red blood cells can be obtained by introducing the parasite into a host, preferably a human, and recovering red blood cells from the infected host when the parasitaemia reaches at least 1%, preferably between 5 and 10%. According to a preferred embodiment, the parasitized red blood cells are recovered from a human host of blood group O and negative Rh.
• the parasitized red blood cells are obtained by ex vivo infection of human red blood cells, preferably red blood cells of blood group O and negative Rh.
• cultures of parasitized red blood cells can be synchronized so as to obtain, for the most part, merozoites, trophozoites or intra- erythrocytic schizonts.
• the methods of ex vivo culturing of Plasmodium parasites are well known to those skilled in the art (see, for example, Trager and Jensen, 1976).
• Anti-coagulants such as heparin may be added to the parasitized red blood cells thus obtained.
• the parasitized red blood cells can be preserved by freezing in the presence of one or more cryoprotectants compatible with an in vivo use, such as, for example, glycerol or dimethylsulfoxide (DMSO).
• the parasitized red blood cells may also be stored by refrigeration at 4 ° C. in a suitable preservation medium, for example SAGM medium ("Adenine Saline"). Mannitol Glucose ”) or a CPD (Citrate Phosphate Dextrose) solution, but for a period not exceeding approximately 45 days.
• the parasites according to the invention are used in the form of sporozoites.
• Sporozoites can be obtained by introducing the parasite into a mosquito host where it will multiply. The sporozoites are then recovered from the salivary glands of infected mosquitoes. The sporozoites thus obtained can be stored by freezing, for example in liquid nitrogen, before being thawed for live injection into a host. Alternatively, after recovery from the salivary glands of the mosquitoes, the sporozoites can be preserved by lyophilization or refrigeration before administration.
• the administration of the parasite according to the invention in a subject makes it possible, in spite of rapid parasite clearance, to induce in the subject an immunity of several months vis-à-vis infection with a Plasmodium, in particular a Plasmodium chosen from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi, preferably Plasmodium falciparum.
• This immunity may especially be a cross-immunity to infection with a strain of Plasmodium different from that of the parasite used.
• the administration of a parasite according to the invention belonging to a strain which does not cause neuropaludism can lead to cross-immunity with respect to an infection with a Plasmodium strain capable of causing this severe neurological complication.
• the parasite according to the invention can therefore be used for the prevention of malaria and / or cerebral malaria.
• the administration of the parasite according to the invention in a subject makes it possible to induce an immunity of several months vis-à-vis infection with Plasmodium falciparum capable of inducing a cerebral malaria and thus to prevent malaria and / or a neuropaludism induced by this parasite.
• prevention refers to an absence of symptoms or the presence of attenuated symptoms of the disease after contact with the parasite.
• malaria prevention refers to an absence of symptoms or to the presence of attenuated symptoms in the subject treated after contact with a Plasmodium responsible for malaria.
• prevention of cerebral malaria refers to an absence of symptoms or to the presence of attenuated symptoms in the subject treated after contact with a Plasmodium responsible for malaria and capable of inducing a cerebral malaria. This term can also refer to the absence of development of a cerebral malaria or the development of a mild or attenuated cerebral malaria in a Plasmodium-infected person capable of inducing cerebral malaria.
• the present invention relates to an immunogenic composition comprising an immunologically effective amount of a parasite according to the invention, and one or more pharmaceutically acceptable excipients or carriers.
• the parasite included in the composition is as described above.
• the composition comprises a parasite according to the invention in an erythrocytic form, more particularly in the form of merozoites, trophozoites or intra-erythrocytic schizonts or non-intraerythrocytic merozoites, preferably in the form of merozoites, trophozoites or intra-erythrocytic schizonts.
• the composition comprises red blood cells parasitized with the parasite according to the invention and which can be obtained according to the method described above and in the experimental part.
• the parasite included in the composition is in the form of sporozoites as described above.
• the immunogenic composition is capable of inducing in the subject to which it is administered, a response of the immune system against the parasite it contains.
• the term "immunologically effective amount” as used herein refers to an amount of parasites that is sufficient to elicit an immune response in the subject.
• the immunogenic composition is a vaccine against malaria.
• the composition according to the invention is obtained by suspending parasitized red blood cells, merozoites or sporozoites, preferably parasitized red blood cells or sporozoites, as defined above, in one or more pharmaceutically acceptable excipients.
• Excipients may be readily selected by those skilled in the art depending on the form of the parasite, intra- erythrocyte, merozoites or sporozoites, and depending on the intended route of administration. These excipients may especially be selected from the group consisting of sterile water, sterile saline and phosphate buffer. Other excipients well known to those skilled in the art can also be used.
• the excipient used is an isotonic solution ensuring the integrity of the red blood cells until administration of the composition to the subject.
• the composition also comprises at least one anticoagulant such as heparin.
• composition may also be obtained by mixing sporozoites of parasites as defined above with a pharmaceutically acceptable carrier such as, for example, liposomes.
• excipients or carriers used are chosen so as to ensure the integrity of parasitized red blood cells and / or the survival of sporozoites or merozoites.
• the excipients or supports used are chosen so as to ensure the survival of the parasites of the invention, whatever the form used (merozoites, sporozoites or intraperiocytic forms), until the administration of the composition to the subject to be immunized. .
• the subject to be immunized is a mammal, preferably a human.
• composition according to the invention can be administered, for example, parenterally, cutaneously, mucosa, transmucosal or epidermal.
• the composition is formulated to be administered parenterally, particularly subcutaneously, intramuscularly, intravenously or intradermally.
• the parasite is in erythrocyte form, preferably included in red blood cells, and the composition is formulated to be administered parenterally, preferably subcutaneously, intramuscularly, intravenously or intradermally, and most preferably intravenously.
• the parasite is in the form of sporozoites and the composition is formulated to be administered parenterally, preferably subcutaneously, intramuscularly or intradermally, preferably intramuscularly or subcutaneously.
• Methods of administering compositions comprising live sporozoites are well known to those skilled in the art (see, for example, International Patent Application WO 2004/045559 and Hoffman et al., 2010).
• the parasite is in erythrocyte form and included in red blood cells and the composition according to the invention comprises between 10 and 10 6 parasitized red blood cells (GRP) per unit dose, preferably between 100 and 10 6 GRP, particularly preferably between 100 and 10 5 GRP, and most preferably between 100 and 10 4 GRP per unit dose.
• the parasite is in erythrocyte form and included in red blood cells and the composition according to the invention comprises between 10 and 100 parasitized red blood cells (GRP) per unit of dose.
• the parasite is in the form of merozoites non intraerythrocytic and the composition of the invention comprises between 10 2 and 10 7 merozoites per unit dose, preferably between 10 3 and 10 5 merozoites per unit dose.
• the parasite is in the form of sporozoites and the composition according to the invention comprises between 10 3 and 10 7 sporozoites per unit dose, preferably between 10 4 and 10 5 sporozoites per unit dose.
• the dose to be administered can be easily determined by those skilled in the art taking into account the physiological data of the subject to be immunized such as its age or immune status, the degree of immunity sought, the number of doses administered and the route of administration. administration used.
• the dose to be administered may also vary according to the mode of conservation of the parasites.
• composition according to the invention may comprise one or more strains of parasites according to the invention.
• the composition comprises at least one strain of Plasmodium falciparum and a strain of Plasmodium vivax in which the function of the hmgb2 gene is inactivated.
• composition according to the invention may also comprise one or more other parasites genetically attenuated Plasmodium genus.
• These parasites may, for example, exhibit alteration or inactivation of purine nucleoside phosphorylase, nucleoside transporter 1, UIS3, UIS4, p52 or p36, or may be attenuated parasites obtained by irradiation.
• These parasites preferably belong to a strain selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
• composition according to the invention may also comprise one or more immunological adjuvants.
• immunological adjuvants include, but are not limited to, adjuvants of the type muramylpeptide such as N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and its derivatives; trehalose dimycolate (TDM); lipopolysaccharide (LPS); monophosphoryl lipid A (MPL); carboxymethylcellulose; the complete adjuvant of Freund; incomplete Freund's adjuvant; adjuvants of "oil-in-water” emulsion type optionally added with squalene or squalane; mineral adjuvants such as alum, aluminum hydroxide, aluminum phosphate, potassium phosphate or calcium phosphate; bacterial toxins such as the cholera toxin subunit B, the inactivated form of the per
• composition according to the invention may comprise one or more immunological adjuvants selected from the group consisting of CpG oligodeoxynucleotides and mineral adjuvants, in particular alum, and a combination thereof.
• the invention relates to the use of a living parasite belonging to the genus Plasmodium in which the function of the hmgb2 gene is inactivated for the preparation of a vaccine composition against malaria or cerebral malaria.
• the invention also relates to a method for producing a vaccine composition against malaria or cerebral malaria according to the invention.
• the method comprises the step of mixing infected red blood cells with a live parasite according to the invention, with one or more pharmaceutically acceptable excipients or carriers.
• the red blood cells are human red blood cells obtained from a host of blood group O and Rh negative.
• the method comprises the step of mixing living non-intraerythrocyte merozoites of a parasite according to the invention with one or more pharmaceutically acceptable excipients or carriers.
• the method comprises the step of mixing live sporozoites of a parasite according to the invention with one or more pharmaceutically acceptable excipients or carriers.
• the parasites in the form of parasitized red blood cells, merozoites or in the form of sporozoites, are also mixed with one or more immunological adjuvants.
• immunological adjuvants may be as defined above.
• the method may further comprise a prior step comprising obtaining said parasitized red blood cells, said merozoites or said sporozoites, for example using the methods described above.
• composition or vaccine obtained may be stored before administration, for example frozen or refrigerated if it contains parasitized red blood cells, or frozen, refrigerated or lyophilized if it contains sporozoites or merozoites.
• the composition or the vaccine obtained is stored frozen before administration.
• a suitable diluent is added to the lyophilisate before administration, such as, for example, sterile water or sterile saline, preferably sterile saline.
• the invention in another aspect, relates to a method for immunizing a subject against malaria, comprising administering an immunogenic composition or vaccine according to the invention to said subject.
• the subject is a mammal, more particularly a human.
• the method comprises administering a vaccine according to the invention to said subject.
• the method comprises the administration of a single dose of the immunogenic composition or of the vaccine according to the invention.
• the method comprises the administration of a plurality of doses of the immunogenic composition or of the vaccine according to the invention. Immunization of the subject can be obtained after administration of 2 to 6 doses. Preferably, a delay of about 4 to 8 weeks, preferably about 6 to 8 weeks, is observed between each administration.
• the inventors have demonstrated that the administration of a single dose comprising 10 5 parasitized red blood cells with a parasite according to the invention made it possible to obtain a sterile immunity of at least 6 months in a mammal.
• the method comprises the administration of a first dose of the immunogenic composition or of the vaccine according to the invention followed by the administration of a second dose about six months to one year later.
• the doses comprise between 10 and 10 6 , preferably between 100 and 10 6 , parasitized red blood cells, between 10 2 and 10 7 merozoites or between 10 3 and 10 7 sporozoites. In a preferred embodiment, the doses comprise about 10 3 parasitized red blood cells. According to another preferred embodiment, the doses comprise between 10 and 100 parasitized red blood cells.
• the subject's immunity to malaria or cerebral malaria may be complete or incomplete.
• incomplete immunity the severity of the symptoms of the disease reported in an immunized subject will be reduced compared to those observed in a non-immune subject.
• total immunity the immune subject will not report any symptoms of the disease after contact with the parasite.
• the immunity obtained by administering the composition according to the invention is a sterile immunity. This means that the development of the administered parasites is very altered and that about 2 to 4 weeks after administration, the parasites are no longer detected in the peripheral blood of the subject.
• the invention also relates to a method for immunizing a subject against malaria, comprising administering a parasite according to the invention in the form of sporozoites to said subject through mosquito bites infected with said parasite.
• the inventors have recently shown that the P. berghei HMGB2 protein is involved in the establishment of experimental neuropaludism (NPE) in a model of C57BL / 6 mice infected with the P. berghei ANKA parasite. (i3 ⁇ 4ANKA).
• NPE experimental neuropaludism
• the inactivation of the hmgb2 gene in i3 ⁇ 4ANKA without hindering the development of the parasite in C57BL / 6 mice, nevertheless causes a delay in the establishment of the NPE or even its complete abolition in 65% of cases.
• the administration of recombinant proteins HMGB2 to mice previously infected with i3 ⁇ 4ANKA Ahmgb2 was able to restore the development of neuropaludism.
• mice used are C57BL / 6 mice (Charles River Laboratories). These mice are sensitive to experimental neuropaludism (NP-S).
• the P. berghei ANKA (i3 ⁇ 4ANKA) parasite induces the death of C57BL / 6 mice by neuropaludism in 7 days +/- 1.
• This parasite has a GFP label under the control of the eefl promoter (Thaty and Ménard, 2002 ).
• P. berghei parasite NK65 (i3 ⁇ 4NK65) (MRA-268) (from Sa et al., 2009) induced death of C57BL / 6 mice by hyper-parasitaemia within 20 to 25 days after infection. On the other hand, this parasite does not cause neuropaludism.
• RH1 meaning 5 'CGATGCGGGCCC AAAAAGGTAAAT ATGAAAAAGAAAGGTT3'
• RH1 an Apal restriction site at 5 'UTR and a Smal restriction site at 3' UTR
• RH2 a NotI restriction site at 5 'UTR and an Ascl restriction site at 3'UTR of the sequence. These restriction sites allow the insertion of fragments
• the 5 'and 3' untranslated regions RH1 and RH2 were then inserted into a pBC SK-Hudhfr vector (Stratagene) containing the human dihydrofolate reductase gene (hudhfr) under the control of the 5'UTR efla promoter and the dhfr / ts (dhfr / thymidilate synthase) at 3'UTR.
• RH1 was inserted in 5'UTR of the Hudhfr cassette and RH2 in 3'UTR.
• the 2 RH1 and RH2 regions matched with the complementary sequences of the hmgb2 gene in the P. berghei genome, allow a double homologous recombination at this gene and, therefore, its deletion.
• Hudhfr confers resistance to pyrimethamine and mutant parasites were selected using this drug.
• the GRPs culture was centrifuged for 10 min at 1500 rpm and the pellet was then taken up in 35 ml of complete culture medium in a 50 ml conical bottom tube.
• Ten milliliters of 50% Nycodenz (Lucron Bioproducts, 1 ⁇ 2 dilution in 1X PBS) were then gently added to the GRPs culture to create a density gradient at the bottom of the tube. Twenty minutes of centrifugation at 450 g, TA, without brake, resulted in the purification of GRPs which then form a brown ring in the middle of the tube.
• the brown ring of GRPs was removed (-20 ml) then washed with 20 ml of complete culture medium. After 8 min of centrifugation at 450 g, the pellet of mature schizonts was finally taken up in 3 ml of complete culture medium which was then distributed in 3 1.5 ml Eppendorf tubes. This operation had to be done in a very delicate way because the schizonts are fragile. At this stage, the amount of schizonts obtained was sufficient to achieve up to 10 independent transfections.
• the pellet of schizonts in each tube was then taken up in 100 ⁇ l of the electroporation buffer (Nucleofector Solution 88A6, AMAXA) with 5 ⁇ g of each of the plasmid constructs obtained (pBC- 5'RHlH " ⁇ i / z / r3'RH2 for pbhmgbl or pbhmgbl), previously digested with ApaI and AscI.
• the parasites were transfected using the U33 program of the electroporator, Nucleofector AMAXA.
• the electric shock induces the rupture of the mature schizont membrane and the merozoites thus released can internalize the linearized plasmids.
• Fifty microliters of complete culture medium were added immediately to the contents of the cuvette and two 3-week-old Swiss mice were immediately infected with 100 ⁇ g each of transfected merozoites.
• the selection drug pyrimethamine
• the pyrimethamine solution was prepared as follows: 70 mg pyrimethamine (Sigma Aldrich), 10 ml DMSO, qsp 1 L water, pH 3.5-5.5.
• the C57BL / 6 mice were infected, intraperitoneally with 200 ⁇ l each, with one ampoule containing 10 7 GRPs / 200 ⁇ l.
• parasitaemia reached 1-5% (5-6 days after infection)
• approximately 100 ⁇ of blood was collected from the animal's cheek in a tube containing heparin. This blood was washed twice with 1 ml of cold PBS and centrifuged for 5 min at 3800 rpm. After dilution to 1/1000 and counting cells, C57BL / 6 mice were infected with 10 5 GRPs. Measuring parasitaemia
• Parasitaemia is analyzed by microscopic counting of parasitized red blood cells (RBCs) in stained smears.
• the blood is taken from infected mice, by intracardiac puncture (anesthetized animal) when the parasitaemia reaches between 3 and 10% (5 to 6 days after infection with 10 7 parasitized red blood cells) in a tube containing heparin and still stored in ice until freezing in liquid nitrogen in the following cryopreservant mixture: 1 vol. Pure glycerol (Sigma Aldrich) + 9 vol Alsever's Solution (Sigma Aldrich).
• the stabilates are carried out with 1 volume of the collected blood to which 2 volumes of the cryopreservant mixture are added. The aliquots are frozen in liquid nitrogen (about 300 ⁇ l per mouse) and then stored at -80 ° C.
• Ampoules as previously, the blood is collected by intracardiac puncture but, when the parasitaemia reaches between 1 and 5%. This blood is washed several times with cold PBS and centrifuged for 5 min at 3800 rpm. Then ampoules of 10 7 GRPs / 200 ⁇ are made in a cryopreservant mixture see previous paragraph after counting the number of parasitized red blood cells.
• the mutated parasite was no longer detected in blood smears while parasite development wilderness continued until death by hyper-parasitaemia about 25 days after infection. Indeed, while the wild parasite continues to develop, infection with i3 ⁇ 4NK65 Ahmgb2 induces an immune response in the C57BL / 6 mouse that alters the development of this parasite. Several independent mutated clones were obtained showing the same clearance kinetics during their development in C57BL / 6 mice.
• Figure 2 shows the results of an experiment with three successive infection tests. All mice primed with i3 ⁇ 4NK65 Ahmgb2 survived the three infection tests with continued sterile protection for at least 7 months. At each challenge, the inventors verified that the development of the wild-type parasite i3 ⁇ 4NK65 in naive mice continues until about 25 days when the mice begin to die from hyper-parasitaemia and that the development of the wild-type 3 ⁇ 4ANKA parasite in naive mice continues until about 7 days when the mice die of cerebral malaria (the witnesses of the pathogenicity of the parasites and the age of the mice).

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