WO2016046321A1 - Procédé pour le traitement d'un cancer et de maladies infectieuses - Google Patents

Procédé pour le traitement d'un cancer et de maladies infectieuses Download PDF

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WO2016046321A1
WO2016046321A1 PCT/EP2015/071997 EP2015071997W WO2016046321A1 WO 2016046321 A1 WO2016046321 A1 WO 2016046321A1 EP 2015071997 W EP2015071997 W EP 2015071997W WO 2016046321 A1 WO2016046321 A1 WO 2016046321A1
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gondii
antigen
gra6
cancer
gra2
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PCT/EP2015/071997
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English (en)
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Nicolas Blanchard
Jodie LOPEZ
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paul Sabatier Toulouse Iii
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2016046321A1 publication Critical patent/WO2016046321A1/fr

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    • 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
    • C07K14/45—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
    • A61K39/012—Coccidia antigens
    • 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
    • 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
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant 1) expresses one or more chimeric GRA6 proteins with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • Plasmodium yoelii the malaria-causing agent, was found to increase the cytotoxic activity of Natural Killer cells (NK) and the maturation of Dendritic Cells (DC) and to inhibit the growth of lung tumors [Chen L. et al, 2011].
  • Injection of genetically attenuated T. gondii, the parasite responsible for toxoplasmosis, in B16F10 melanoma was reported to stimulate both the innate (NK) and adaptive responses (CD8) and to result in tumor regression [Baird J. et al, 2013a].
  • the adjuvant effect of T. gondii was even powerful enough to reverse the immunosuppression observed in a model of aggressive ovarian tumor [Baird J.
  • a second potential advantage of intracellular parasites is that they may be genetically modified to express a tumor Ag, thereby creating a tumor vaccine vector able to licence T cells to specifically kill tumor cells.
  • a tumor vaccine vector able to licence T cells to specifically kill tumor cells.
  • One example is the administration of Leishmania tarentolae parasites expressing a papillomavirus protein, which induced an effective adaptive response against tumors expressing this Ag [Salehi M. et al, 2012].
  • the recent data presented here on the mechanisms controlling immunogenicity of T. gondii Ags strongly suggest that T. gondii could represent a parasite of choice for a CD8 T cell-boosting vaccine.
  • Toxoplasma gondii (T. gondii) is an obligate intracellular parasite able to chronically infect any warm-blooded animal. In the human, T. gondii is responsible for severe fetal abnormalities and for encephalitis in immunocompromised individuals. Following host cell invasion, T. gondii multiplies within a parasitophorous vacuole segregated from the cytosol by a limiting membrane, which thus lies at the interface with the host cell. Active entry and vacuole formation result from the coordinated secretion of parasite secretory organelles called micronemes, rhoptries and dense granules [Sibley LD et al, 2011].
  • IVN IntraVacuolar Network
  • the IVN may be involved in virulence [Mercier C et al, 1998] by helping to route parasite rhoptry effectors released in the host cytosol back to the vacuole membrane and thus preventing vacuole destruction by immunity-related GTPases [Reese ML et al., 2009 and Niedelman W et al, 2012]. More recently, the IVN has been implicated in a novel pathway of host cytosolic material ingestion by the parasite [Dou Z et al, 2014]. Yet, both the exact molecular bases underlying IVN biogenesis, as well as its exact function remain ill-defined. Detection of T.
  • gondii intrusion by CD8 T cells relies on the recognition of short antigenic peptides (8 to 10 amino acids) presented by Major Histocompatibility Complex (MHC) class I molecules at the surface of infected cells.
  • MHC Major Histocompatibility Complex
  • the generation of antigenic peptides from endogenous sources typically involves processing by cytosolic proteases, transport into the endoplasmic reticulum (ER), trimming by ER-resident aminopeptidases and loading onto peptide-receptive MHC I molecules [Blum JS et al, 2013]. In the case of T.
  • T. gondiioi one or several tumor antigens by T. gondii, which could, following vaccination, boost the CD8 responses and potentiate tumor rejection or elimination of an infectious pathogen.
  • the invention relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant 1) expresses one or more chimeric GRA6 proteins with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • the present invention relates to an attenuated mutant of Toxoplasma gondii which 1) expresses one or more chimeric GRA6 proteins with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • the attenuated mutant of Toxoplasma gondii may be used for the prevention or the treatment of cancer or of infectious diseases.
  • the present invention also relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant 1) expresses one or more chimeric GRA6 proteins with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • GRA6 for "Dense Granule Protein 6" denotes an antigenic protein of Toxoplasma gondii which induces a strong CD8 response. GRA6 is stored in secretory compartments of the parasite (called dense granules) and secreted into the vacuole after host cell invasion.
  • An exemplary sequence for the GRA6 gene and protein of type II T gondii is deposited in ToxoDB.org version 11.0 under the accession number TGME49 275440.
  • An exemplary sequence for the GRA6 gene and protein of type I T gondii is deposited in ToxoDB.org version 11.0 under the accession number TGGT1 275440.
  • GAA2 for "Dense Granule Protein 2" denotes a protein of Toxoplasma gondii which is implicated in the synthesis of the nanotubular network in Toxoplasma gondii vacuole.
  • An exemplary sequence for the GRA2 gene and protein of type II T gondii is deposited in ToxoDB.org version 11.0 under the accession number TGME49 227620.
  • An exemplary sequence for the GRA2 gene and protein of type I T gondii is deposited in ToxoDB.org version 11.0 under the accession number TGGT1 227620.
  • the term "expresses one or more chimeric GRA6 proteins with an antigen at its C-terminus” denotes the protein GRA6 with an antigenic peptide or polypeptide encoded at the C-terminal extremity of the GRA6 protein.
  • the antigen is linked to the GRA6 protein of any lineage of T. gondii, after the very last C-terminal amino acid (F or Y), or as a replacement of the last 10 amino acid sequence, i.e. after the leucine at position 214, or directly following the GRA6 transmembrane domain (aa 153-171). In this latter case, the rest of the protein (C-terminal domain) is removed.
  • the attached antigenic peptide or polypeptide is preceded by a leucine residue to facilitate N-terminal processing.
  • Expression of more than one chimeric GRA6 protein can be envisaged using bicistronic expression from the same expression vector, or using distinct vectors with identical or different selectable markers.
  • the attenuated mutant of Toxoplasma gondii may express one or more than one chimeric GRA6 proteins with an antigen at its C-terminus.
  • the attenuated mutant of Toxoplasma gondii may express one chimeric GRA6 protein with a specific antigen at its C-terminus and one or several other chimeric GRA6 proteins with a different specific antigen at its C-terminus.
  • the invention relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant 1) expresses one chimeric GRA6 protein with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • the invention relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant 1) expresses two or more chimeric GRA6 proteins with an antigen at its C-terminus and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • a knockout or loss of function mutation of the gene coding for the GRA2 protein denotes a deletion of the GRA2 gene or a deletion of the three most important amphipathic helices of the gene (helix al : aa 70-92 ; helix a2: aa 95-110 ; helix a3 : aal 19-139) which induce a loss of function of the protein GRA2 and/or a loss of the expression of GRA2.
  • the knockout or loss of function mutation includes a substitution, deletion, or insertion at GRA2 gene which decreases or abolishes the activity of the GRA2 protein encoded by said gene.
  • the attenuated mutant of Toxoplasma gondii is an attenuated mutant of Toxoplasma gondii which has a knockout or loss of function mutation of the gene coding for the protein micl and mic3 (Micl-3KO) such as described in WO2005/072754; Mevelec et al 2010 and Odile et al 2005.
  • the present invention also relates to an attenuated mutant of Toxoplasma gondii for use in the prevention or the treatment of cancer or of infectious diseases in a subject in need thereof wherein the attenuated mutant has a knockout or loss of function mutation of the gene coding for the protein micl and mic3.
  • An attenuated mutant of the present invention can be generated using any suitable method conventionally employed for producing gene knockout mutants of T gondii.
  • the mutant can be obtained by the single cross-over integration, e.g., as disclosed by Fox & Bzik ((2002) Nature 415(6874):926-9) or using a double-crossover gene replacement, e.g., as disclosed by Mercier, et al. ((1998) Infect. Immun. 66:4176-82). See also Wang, et al. (2002) Mol. Biochem. Parasitol.
  • the generation of a mutant T gondii includes isolating the nucleic acid molecule of interest from T gondii; replacing, mutating, substituting or deleting all or a portion (e.g., one or more bp) of the gene to disrupt the promoter, regulatory sequence(s) and/or coding region of the protein; and integrating the disrupted molecule (e.g., via single- or double-crossover homologous recombination events) into the genome of T gondii.
  • a knockout mutant Upon selection, i.e., marker protein expression or genomic DNA analysis, a knockout mutant is obtained.
  • the selectable marker is selected for by positive and negative selection (e.g., HXGPRT), such that the selectable marker can be easily deleted from the targeted locus by homologous recombination and, upon negative selection, recovered for use again in a sequential process of positive and negative selection to create strains harboring multiple gene knockouts or replacements.
  • positive and negative selection e.g., HXGPRT
  • Disruption of all or a portion of a gene of interest can be achieved by, e.g., replacing the coding sequence with a nucleic acid molecule encoding selectable marker, replacing the coding sequence with a nucleic acid molecule encoding an exogenous protein, substituting the promoter with a mutated promoter which can no longer be recognized by T. gondii transcription proteins (i.e., a promoter mutation), etc.
  • subsequent restriction endonuclease digestion and Southern blot analysis of the mutant T. gondii genomic DNA can be used to confirm the knockout.
  • any suitable marker-encoding nucleic acid can be used to identify a T. gondii which has been transformed so long as it can be phenotypically detected in the mutant strain.
  • Suitable marker proteins include, but are not limited to, positive and negative selectable markers such as HXGPRT, thymidine kinase, hygromycin resistance, cytosine deaminase, DHFR (dihydro folate reductase), bleomycin, chloramphenicol acetyl transferase, or combinations thereof. It is contemplated that the nucleic acid molecule encoding the marker protein can be used to replace or substitute all or a portion of the promoter or coding sequence of the locus of interest to generate a knockout or mutant.
  • the attenuated mutant T. gondii of the invention is a ⁇ - irradiated attenuated mutant strain of T. gondii.
  • the use of ⁇ irradiation to attenuate T. gondii is described in the art (Dubey, et al, 1998 Int. J. Parasitol. 28:369-75; Kook, et al, 1995 Korean J. Parasitol. 33: 173-8 and Hiramoto et al, 2002].
  • gamma-irradiated tachyzoites maintained metabolic function and the ability to invade cells and preserved protein and nucleic acid synthesis, while their replication was blocked.
  • Irradiated tachyzoites also induced a humoral and a cellular immune response.
  • the attenuated mutant maintain the ability to invade cells, including DC and myeloid cells, to provide optimal antitumor or anti-infectious responses.
  • an antigen refers to a molecule which is capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes.
  • An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes).
  • the antigen according to the invention is an antigenic peptide.
  • the antigen may be a "Tumor antigen".
  • tumor antigen refers to an antigen that is characteristic of a tumor tissue.
  • Example of a tumor antigens include, but are not limited to antigen prostatic acid phosphatise (see WO 2004026238), MART peptide T (melanoma antigen) mesothelin, CEA, p53, Her2/neu, ErB2, melan A, MAGE antigens, nm23, BRACA1 , and BRACA2.
  • the tumor antigen is expressed by T. gondii, secreted into the parasite vacuole and eventually into the cytosol of the mammalian host cell.
  • the T. gondii- expressed tumor antigen subsequently enters the mammalian antigen presenting cell's (APC) antigen processing and presenting pathway as a substrate for generation of class I and class II peptides which generate CD8 and CD4 T cell responses.
  • APC mammalian antigen presenting cell's
  • the attenuated mutant of the invention harbors nucleic acid molecules encoding one or more tumor antigens.
  • the basic criteria for tumor antigen expression are that the gene is a non- T. gondii gene or coding sequence and the gene or coding sequence is able to be expressed directly or indirectly from a recombinant molecule in a T. gondii cell.
  • the promoter employed is recognizable by T. gondii.
  • the promoter promotes transcription of the protein coding sequence when the T. gondii is inside mammalian cells.
  • particular embodiments embrace the use of a T. gondii promoter.
  • Known promoter and other regulatory elements which can be operably linked to the coding sequence of an exogenous protein of interest so that the exogenous protein is expressed in T. gondii include, but are not limited to, sequences from the T. gondii SAG1 gene (Striepen, et al. (1998) Mol. Biochem. Parasitol. 92(2):325-38) or the T. gondii NTPase gene (Robibaro, et al. (2002) Cellular Microbiol. 4: 139; Nakaar, et al. (1998) Mol. Biochem. Parasitol. 92(2):229-39).
  • suitable regulatory sequences can be obtained by known trapping techniques. See, e.g., Roos, et al. (1997) Methods 13(2): 112-22.
  • Promoters of use in accordance with the present invention can also be stage-specific promoters, which selectively express the exogenous protein(s) or antigen(s) of interest at different points in the obligate intracellular T. gondii life cycle.
  • an endogenous promoter can be used to drive expression of the exogenous protein or antigen by, e.g., site-specific integration at the 3 end of a known promoter in the T. gondii genome.
  • the term "attenuated" refers to a weakened and/or less vigorous strain of T. gondii.
  • the attenuated mutant of the invention is capable of stimulating an immune response and creating immunity but not causing illness.
  • Attenuation can be achieved by conventional methods including, but not limited, to ⁇ -irradiation or the generation of a pyrimidine auxotroph.
  • a pyrimidine auxotroph of the invention can be generated by disrupting mechanisms for pyrimidine acquisition including, mutating proteins involved in pyrimidine synthesis along with those of pyrimidine salvage (e.g., enzymes or transporters).
  • pyrimidine auxotrophs can be produced by knocking out or mutating one or more of CPSII (carbamoyl phosphate synthetase II; Gene loci ID 583.m05492), OMPDC (orotidine 5 ' - monophosphate decarboxylase; Gene loci ID 55.m04842), OPRT (orotate phosphoribosyltransferase; Gene loci ID 55.m04838), DHO (dihydroorotase; Gene loci ID 83.m00001), aspartate transcarbamylase (ATC), dihydroorotase dehydrogenase (DHOD), uridine phosphorylase (UP), uracil phosphoribosyltransferase, purine nucleoside phosphorylase (e.g., PNP), or a nucleobase/nucleoside transporter of pyrimidine bases or nucleosides (e.g., P
  • any single knockout or combination of knockouts is contemplated to achieve an attenuated strain.
  • the present embraces an attenuated strain or vaccine strain constructed by a single knockout in any of the six de novo pyrimidine biosynthetic genes (CPS, ATC, DHO, DHOD, OPRT or OMPDC), knockout of two or more genes of the de novo pyrimidine synthetic pathway, or knockout of a de novo pyrimidine synthesis gene in combination with a knockout in a pyrimidine salvage gene (e.g., coding for enzymes UP, PNP, or uracil phosphoribosyltransferase) and/or in combination with a knockout of a nucleobase/nucleoside transporter of pyrimidine bases or nucleosides.
  • CPS de novo pyrimidine biosynthetic genes
  • ATC de novo pyrimidine biosynthetic genes
  • DHO genes of the de novo
  • Such attenuated mutants can be generated by substitution, deletion or insertion as is conventional in the art. It is contemplated that because an attenuated pyrimidine auxotroph of T. gondii (e.g., a CPSII or OMPDC knockout) induces a Thl immune response, any attenuated mutant of T. gondii can be used as a vaccine without the complication of dead host cells and infectious dissemination of T. gondii in the host.
  • an attenuated pyrimidine auxotroph of T. gondii e.g., a CPSII or OMPDC knockout
  • any attenuated mutant of T. gondii can be used as a vaccine without the complication of dead host cells and infectious dissemination of T. gondii in the host.
  • the attenuated mutant of Toxoplasma gondii can be produced from a virulent type I strain such as RH, a type II strain as well as a type III strain for instance by using a CRISPR/Cas9-based genome editing method (Shen B et al Mbio 2014; Sidik SM et al PloS One 2014).
  • the attenuated mutant of T. gondii according to the invention can be used to express any exogenous protein one would want to express within a mammalian host cell.
  • therapeutic peptides or proteins e.g., therapeutic antibodies (e.g., Trastuzumab) proteins (e.g., interferons, blood factors, insulin, erythropoietin, and blood clotting factors), or enzymes (e.g., asparaginase, catalase, lipase, and tissue plasminogen activator) used in the treatment of diseases or conditions.
  • therapeutic antibodies e.g., Trastuzumab
  • proteins e.g., interferons, blood factors, insulin, erythropoietin, and blood clotting factors
  • enzymes e.g., asparaginase, catalase, lipase, and tissue plasminogen activator
  • proteins are routinely expressed in other systems, e.g., yeast, mammalian cells lines, bacteria or insect cells, such that one skilled in the art could readily obtain nucleic acids encoding such proteins and express them in a mutant T. gondii.
  • the cancer may be selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g.
  • bile duct cancer e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibro
  • breast cancer e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angio follicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g.
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g.
  • rhabdomyosarcoma embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).
  • skin cancer e.g. melanoma, nonmelanoma skin cancer
  • stomach cancer testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma
  • infectious diseases may be a disease caused by a virus, a bacterium, a parasite or a fungus.
  • infectious diseases may be a Chikungunya virus infection, a Smallpox infection, a severe acute respiratory syndrome (SARS) infection, Hepatitis C virus (HCV) infection, Epstein-Barr Virus (EBV) infection, Hepatitis B virus (HBV) infection, Human immunodeficiency virus (HIV) infection, cytomegalovirus (CMV) infection, varicella zoster virus infection, an herpes infection avian influenza, Human Herpes Virus 1 (HHV-1) infection, HHV-2 infection, HHV-6 infection, HHV-8 infection, coxsackie virus B4 infection, influenza A and B viruses infection, a Measles virus infection, a Rubella virus infection, a Human Papilloma Virus (HPV) infection.
  • SARS severe acute respiratory syndrome
  • HCV Hepatitis C virus
  • EBV Epstein-Barr Virus
  • HBV Hepatitis B virus
  • HIV Human immunodeficiency virus
  • CMV cyto
  • infectious diseases may be caused by a bacterium like
  • Mycobacterium tuberculosis Mycobacterium leprae or lepromatosis, Staphylococcus aureus, Streptococcus A, Helicobacter pylori, Chlamydia trachomatis, Mycoplasma pneumoniae, Haemophilus influenza, Borrelia burgdorferi, Bartonella Hensalae.
  • infectious diseases may be caused by a parasite like Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania major or Leishmania donovani.
  • the antigen according to the invention may be a virus-specific antigen such as a CMV specific antigen, an EBV specific antigen, a HCV specific antigen, a HBV specific antigen, a Varicella zoster virus specific antigen, a Rubella virus specific antigen or a Measles virus specific antigen.
  • a virus-specific antigen such as a CMV specific antigen, an EBV specific antigen, a HCV specific antigen, a HBV specific antigen, a Varicella zoster virus specific antigen, a Rubella virus specific antigen or a Measles virus specific antigen.
  • a method for preventing or treating cancer or infectious diseases by administering an effective amount in a subject in need thereof of an attenuated mutant of Toxoplasma gondii wherein the attenuated mutant 1) expresses one or more chimeric GRA6 proteins with an antigen at its C-Terminal part and 2) has a knockout or loss of function mutation of the gene coding for the GRA2 protein.
  • the attenuated mutant of Toxoplasma gondii of the invention may be used for the preparation of a vaccine composition.
  • the present invention also provides a vaccine composition comprising the attenuated mutant of Toxoplasma gondii according to the invention.
  • the vaccine composition may comprise attenuated mutant of Toxoplasma gondii with other antigens.
  • the vaccine composition comprising the attenuated mutant of Toxoplasma gondii according to the invention may be used in the prevention or the treatment of cancer or of infectious diseases.
  • a "vaccine composition” once it has been administered to a subject or an animal, elicits a protective immune response against said one or more antigen(s) that is (are) comprised herein. Accordingly, the vaccine composition of the invention, once it has been administered to the subject or the animal, induces a protective immune response against, for example, a microorganism, or to efficaciously protect the subject or the animal against infection.
  • the vaccine composition may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
  • auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like.
  • Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.
  • This pharmaceutical composition can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e. g. human serum albumin) suitable for in vivo administration.
  • additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e. g. human serum albumin) suitable for in vivo administration.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • adjuvant refers to a substance that enhances, augments or potentiates the host's immune response to an antigen, e.g., an antigen that is part of a vaccine.
  • antigen e.g., an antigen that is part of a vaccine.
  • Non-limiting examples of some commonly used vaccine adjuvants include insoluble aluminum compounds, calcium phosphate, liposomes, VirosomesTM, ISCOMS®, microparticles (e.g., PLG), emulsions (e.g., MF59, Montanides), virus-like particles & viral vectors.
  • PolylCLC (a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L- lysine double-stranded R A), which is a TLR3 agonist, is used as an adjuvant in the present invention. It will be understood that other TLR agonists may also be used (e.g. TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR9 agonists), or any combinations or modifications thereof.
  • adjuvants examples include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • MTP-PE N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine
  • MTP-PE N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine
  • adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA.
  • immune modulating substances such as lymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma] inducers such as poly I:C can be used in combination with adjuvants described herein.
  • Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or vectors encoding such adjuvants.
  • Adjuvants that may be used in accordance with embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, ⁇ -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP
  • CGP MTP-PE
  • MPL monophosphoryl lipid A
  • MPL monophosphoryl lipid A
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated.
  • MHC antigens may even be used.
  • Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed
  • TLR4 agonist denotes a compound or a molecule which bind the Toll-like receptor 4 and active it.
  • a TLR4 agonist may be selected from the group consisting of Ethanol, Morphine-3-glucuronide, Morphine, Oxycodone, Levorphanol, Pethidine, Glucuronoxylomannan from Cryptococcus, Fentanyl, Methadone, Buprenorphine, Lipopolysaccharides (LPS), Carbamazepine, Oxcarbazepine.
  • the TLR4 agonist according to the invention is selected from the group consisting of the LPS or monophosphoryl lipid A (MPL).
  • TLR4 agonists are known in the art, including Monophosphoryl lipid A (MPLA), in the field also abbreviated to MPL, referring to naturally occurring components of bacterial lipopolysaccharide (LPS); refined detoxified endotoxin.
  • MPL is a derivative of lipid A from Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin). While LPS is a complex heterogeneous molecule, its lipid A portion is relatively similar across a wide variety of pathogenic strains of bacteria.
  • MPL used extensively as a vaccine adjuvant, has been shown to activate TLR4 (Martin M. et al, 2003. Infect Immun.
  • TLR4 agonists also include natural and synthetic derivatives of MPLA, such as 3-de-O-acylated monophosphoryl lipid A (3D- MPL), and MPLA adjuvants available from Corixa Corporation (Seattle, Wash.; see US Patents 4,436,727; 4,436,728; 4,987,237; 4,877,611; 4,866,034 and 4,912,094 for structures and methods of isolation and synthesis).
  • a structure of MPLA is disclosed in US 4,987,237.
  • Nontoxic diphosphoryl lipid A may also be used, for example OM-174, a lipid A analogue of bacterial origin containing a triacyl motif linked to a diglucosamine diphosphate backbone.
  • Another class of useful compounds are synthetic lipid A analogue pseudo-dipeptides derived from amino acids linked to three fatty acid chains (see for example EP 1242365), such as OM- 197-MP-AC, a water soluble synthetic acylated pseudo-dipeptide (C55H107N4O12P).
  • Nontoxic TLR4 agonists include also those disclosed in EP 1091928, PCT/FR05/00575 or PCT/IB2006/050748.
  • TLR4 agonists also include synthetic compounds which signal through TLR4 other than those based on the lipid A core structure, for example an amino alkyl glucosaminide 4-phosphate (see Evans JT et al. Expert Rev Vaccines. 2003 Apr;2(2):219-29; or Persing et al. Trends Microbiol. 2002;10(10 Suppl):S32-7. Review).
  • TLR9 agonist denotes a compound or a molecule that binds the Toll-like receptor 9 and actives it.
  • a TLR9 agonist may be selected from the group consisting of CpG oligonucleotides (ODN) and its derivatives.
  • the TLR9 agonist is the CpG (ODN).
  • TLR9 agonists include nucleic acids comprising the sequence 5 -CG-3' (a
  • C C is unmethylated.
  • polynucleotide and “nucleic acid,” as used interchangeably herein in the context of TLR9 agonist molecules, refer to a polynucleotide of any length, and encompasses, inter alia, single- and double-stranded oligonucleotides (including deoxyribonucleotides, ribonucleotides, or both), modified oligonucleotides, and oligonucleosides, alone or as part of a larger nucleic acid construct, or as part of a conjugate with a non-nucleic acid molecule such as a polypeptide.
  • a TLR9 agonist may be, for example, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA).
  • TLR9 agonists also encompass crude, detoxified bacterial (e.g., mycobacterial) RNA or DNA, as well as enriched plasmids enriched for a TLR9 agonist.
  • a "TLR9 agonist-enriched plasmid" refers to a linear or circular plasmid that comprises or is engineered to comprise a greater number of CpG motifs than normally found in mammalian DNA.
  • a TLR9 agonist used in a subject composition comprises at least one unmethylated CpG motif.
  • a TLR9 agonist comprises a central palindromic core sequence comprising at least one CpG sequence, where the central palindromic core sequence contains a phosphodiester backbone, and where the central palindromic core sequence is flanked on one or both sides by phosphorothioate backbone-containing polyguanosine sequences.
  • a TLR9 agonist comprises one or more TCG sequences at or near the 5' end of the nucleic acid; and at least two additional CG dinucleotides.
  • the at least two additional CG dinucleotides are spaced three nucleotides, two nucleotides, or one nucleotide apart.
  • the at least two additional CG dinucleotides are contiguous with one another.
  • a TLR9 agonist of the present invention includes, but is not limited to, any of those described in U.S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705, 6,426,334 and 6,476,000, and published US Patent Applications US 2002/0086295, US 2003/0212028, and US 2004/0248837.
  • TLR4 or TLR9 agonists are described in WO 2012/021834, the contents of which are incorporated herein by reference.
  • a variety of substances, used as supplemental antigens, can be added to the vaccine composition.
  • attenuated and inactivated viral and bacterial pathogens, purified macromolecules, toxoids, recombinant antigens, organisms containing a foreign gene from a pathogen, synthetic peptides, polynucleic acids, antibodies and tumor cells can be used to prepare (i) an immunogenic composition useful to induce an immune response in an individual or (ii) a vaccine useful for treating a pathological condition.
  • the vaccine composition of the invention can be combined with a wide variety of antigens to produce a vaccine composition useful for inducing an immune response in an individual or in an animal.
  • the vaccine composition according to the invention further comprises one or more components selected from the group consisting of surfactants, absorption promoters, water absorbing polymers, substances which inhibit enzymatic degradation, alcohols, organic solvents, oils, pH controlling agents, preservatives, osmotic pressure controlling agents, propellants, water and mixture thereof.
  • the vaccine composition according to the invention can further comprise a pharmaceutically acceptable carrier.
  • the amount of the carrier will depend upon the amounts selected for the other ingredients, the desired concentration of the antigen, the selection of the administration route, oral or parenteral, etc.
  • the carrier can be added to the vaccine at any convenient time. In the case of a lyophilised vaccine, the carrier can, for example, be added immediately prior to administration. Alternatively, the final product can be manufactured with the carrier.
  • appropriate carriers include, but are not limited to, sterile water, saline, buffers, phosphate-buffered saline, buffered sodium chloride, vegetable oils, Minimum Essential Medium (MEM), MEM with HEPES buffer, etc.
  • suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ- Amine or NZ-Amine AS.
  • emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil and other standard, metabolizable, nontoxic oils useful for injectables or intranasal vaccines compositions.
  • preservatives can be added to the vaccine composition in effective amounts ranging from about 0.0001% to about 0.1% by weight. Depending on the preservative employed in the formulation, amounts below or above this range may be useful. Typical preservatives include, for example, potassium sorbate, sodium metabisulfite, phenol, methyl paraben, propyl paraben, thimerosal, etc.
  • the vaccine composition of the invention can be formulated as a solution or suspension together with a pharmaceutically acceptable medium.
  • Such a pharmaceutically acceptable medium can be, for example, water, phosphate buffered saline, normal saline or other physiologically buffered saline, or other solvent or vehicle such as glycol, glycerol, and oil such as olive oil or an injectable organic ester.
  • a pharmaceutically acceptable medium can also contain liposomes or micelles, and can contain immunostimulating complexes prepared by mixing polypeptide or peptide antigens with detergent and a glycoside, such as Quil A.
  • Liquid dosage forms for oral administration of the vaccine composition of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubil
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active ingredient(s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the vaccine compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the active ingredient(s) with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active ingredient(s).
  • suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active ingredient(s).
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate
  • Vaccine compositions of this invention suitable for parenteral administration comprise the active ingredient(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions.
  • isotonic agents such as sugars, sodium chloride, and the like in the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • Injectable depot forms are made by forming microencapsule matrices of the active ingredient(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the active ingredient(s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient(s) can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable formulations are also prepared by entrapping the active ingredient(s) in liposomes or microemulsions that are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • the amount of attenuated mutant of Toxoplasma gondii, eventually supplemental antigen and adjuvant composition in the vaccine composition according to the invention are determined by techniques well known to those skilled in the pharmaceutical art, taking into consideration such factors as the particular antigen, the age, sex, weight, species, and condition of the particular animal or patient, and the route of administration.
  • the dosage of the vaccine composition depends notably upon the antigen, species of the host vaccinated or to be vaccinated, etc.
  • the dosage of a pharmacologically effective amount of the vaccine composition will usually range from about 0.01 ⁇ g to about 500 ⁇ g (and in particular 50 ⁇ g to about 500 ⁇ g) of the adjuvant compound of the invention per dose.
  • the amount of the particular antigenic substance in the combination will influence the amount of the adjuvant compound according to the invention, necessary to improve the immune response, it is contemplated that the practitioner can easily adjust the effective dosage amount of the adjuvant compound through routine tests to meet the particular circumstances.
  • the vaccine composition according to the invention can be tested in a variety of preclinical toxico logical and safety studies well known in the art.
  • such a vaccine composition can be evaluated in an animal model in which the antigen has been found to be immunogenic and that can be reproducibly immunized by the same route proposed for human clinical testing.
  • the vaccine composition according to the invention can be tested, for example, by an approach set forth by the Center for Biologies Evaluation and Research/Food and Drug Administration and National Institute of Allergy and Infectious Diseases.
  • the vaccine may be advantageously administered as a unique dose or preferably, several times e.g., twice, three or four times at week or month intervals, according to a prime/boost mode.
  • the appropriate dosage depends upon various parameters.
  • the vaccine composition of the present invention is conveniently administered orally, parenterally (subcutaneously, intramuscularly, intravenously, intradermally or intraperitoneally), intrabuccally, intranasally, or transdermally, intralymphatically, intratumorally, intravesically, intraperitoneally and intracerebrally.
  • parenterally subcutaneously, intramuscularly, intravenously, intradermally or intraperitoneally
  • intrabuccally intranasally
  • transdermally intralymphatically, intratumorally, intravesically, intraperitoneally and intracerebrally.
  • the route of administration contemplated by the present invention will depend upon the antigen.
  • the vaccinal composition of the present invention may be used in human health or animal health.
  • the vaccinal composition may be useful to prevent diseases in human and animal.
  • the invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Association of T. gondii dominant antigen to lipid membranes of the vacuole promotes MHC I presentation by infected cells
  • FIG. 2 In infected bone marrow-derived macrophages (BMM), highly curved membranes of the IVN decrease MHC I presentation of membrane-bound antigen
  • Each dot represents the median value of 3 ratio measurements performed on 3 middle sections of a single vacuole.
  • C57BL/6JxDBA/2 Fl (B6D2) mice were purchased from Janvier (France) and housed under specific pathogen- free conditions. Sex and age-matched (8 to 12 week-old) mice were used. Tachyzoites of parental and transgenic lines from these strains were maintained by serial passages on confluent monolyaers of human foreskin fibroblasts (HFF). Antibodies for flow cytometry were rat anti-CD4 (RM4-5), rat anti-CD8 (53-6.7), mouse anti-IFN- ⁇ (XMG1.2) (eBioscience).
  • tachyzoites were electroporated with linearized plasmid DNA and inoculated in 4 confluent flasks of HFF in order to isolate up to 4 independent clones. The next day, selection was applied and resistant tachyzoites were cloned by limiting dilution. Presence of the transgene was verified by PCR and sequencing from genomic DNA for each clone.
  • HFF HFF were disrupted with a 23-G needle and tachyzoites were lysed in a lysis buffer containing 1% NP-40, 10 mM Tris pH 7.4, 150 mM NaCl, and protease inhibitors (cOmplete EDTA-free, Roche) for 30 min on ice. Lysates were centrifuged for 15 min at 15,000g. Solubilized proteins were reduced in SDS sample buffer, separated by electrophoresis on 12% polyacrylamide gels and transferred to nitrocellulose membranes. Immunologic detection was achieved using horseradish peroxidase-conjugated antibodies. Peroxidase activity was visualized by chemiluminescence and quantified using ChemiDoc camera (Biorad).
  • Bone marrow cells were obtained from B6D2 Fl femurs and tibias.
  • BMDM were differentiated for 7 days in Petri dishes with RPMI supplemented with 20% (vol/vol) FCS and 10% (vol/vol) colony- stimulating factor-containing culture supernatant (purity, about 95% CDl lb+).
  • BMDC were differentiated for 6-8 days with 10% (vol/vol) of granulocyte- macrophage colony- stimulating factor-containing culture supernatant in complete RPMI medium (purity, about 70% CDl lc+).
  • CSF-producing and GM-CSF-producing cells were a gift from R. Vance (UC Berkeley, CA, USA) and C.
  • TCR-mediated stimulation of ⁇ - galactosidase production by the hybridomas was quantified using a chromogenic substrate: chlorophenol red-P-D-galactopyranoside (CPRG, Roche). Cleavage of the CPRG by ⁇ - galactosidase releases a purple product, which absorbance was read at 595 nm with a reference at 655 nm.
  • CPRG chlorophenol red-P-D-galactopyranoside
  • mice were immunized twice 3 weeks apart with 10 6 tachyzoites that were previously ⁇ - irradiated (120 Gy) and filtered through a 3 ⁇ filter (Millipore). Mice were euthanized 4 days after the second immunization.
  • Peritoneal exudate cells PEC
  • Spleens were dissociated into single-cell suspensions in complete RPMI medium (Invitrogen) supplemented with 10% (vol/vol) FCS (Hyclone). Samples were depleted of erythrocytes with ACK lysis buffer (100 ⁇ EDTA, 160 mM NH4C1 and 10 mM NaHC03).
  • Monolayer of HFF grown on Lab-Tek II chamber slides CC2 were infected with T. gondii for 16 to 24 h. After infection, cells were washed in PBS, fixed for 20 min in 3% paraformaldehyde (Electron Microscopy Sciences) in PBS at room temperature (RT) and quenched with PBS 0.1M glycine for 5 min. Primary antibodies were diluted in blocking buffer (PBS, 0.2% BSA, 0.05% saponin) and incubated for lh at RT followed by 3 x 5 min washes.
  • PBS blocking buffer
  • Plasmids were constructed either with traditional ligation or with fusion of a PCR amplicon into an open vector, using the In-Fusion HD Cloning kit (Clontech). A list of all plasmids and their purpose is shown below.
  • GRA6(II) pCG2-5'PstI-3'SalI Mercier et Complement with GRA2(I), TgRH AGRA6(I) al, 1993
  • cotransfected with pDHFR-TSc3 GRA6(II) AGRA2 cotransfected with pDHFR-TSc3 GRA6(II) AGRA2 (Donald and Roos, 1993) for dhfr
  • pBTH/ GRA6(II)/BLE was obtained by PCR-amplifying the GRA6(II) ORF from Pru genomic DNA and cloning the fragment by ligation into the Bglll/Avrll open pBTH vector (van Dooren et al., 2008).
  • pTko.mCherry/GRA2/HPT/GRA2 was generated in two consecutive steps, starting from the pTko.mCherry/HPT plasmid (gift from J. Boothroyd) to create a final vector where the hpt gene was flanked with the 5' and 3' arms of the GRA2(II) gene. 1.5 kb-long flanking sequences were PCR-amplified from Pru genomic DNA and cloned using the XhoI/EcoRI for the 5 ' arm and Hindlll/Nhel for the 3 ' arm.
  • constructs were cloned into pBLUESCRIPT vector using the In-Fusion HD Cloning kit (Clontech).
  • Knock-out clones were complemented by cotransfection of 10 7 cells with 50 ⁇ g of the linearized plasmid pCG2-5'PstI-3'SalI (Mercier et al, 1993) mixed with 5 ⁇ g of the linearized plasmid pDHFR-TSc3 (Donald and Roos, 1993) and selected for pyrimethamine.
  • the plasmid pGRA2/Ble/GRA2 (8.9) (Mercier et al, 1998a) was electroporated into tachyzoites. Phleomycin was added to allow selection and stable clones were isolated by limiting dilution in 96-well micro titer plates.
  • 2 or 3 plasmids of chimeric GRA6 were used.
  • 2xl0 7 CEP.Ahpt.GFP.Luc (Kamau et al., 2011) were cotransfected by electroporation with 50 ⁇ g of the linearized plasmid of interest mixed with 5 ⁇ g of the linearized plasmid pminiHXGPRT (Donald et al., 1996).
  • xanthine and mycophenolic acid stable clones were cloned by limiting dilution in 96-well microtiter plates.
  • HFF cells grown into flat-bottom 6-well plates were infected with parasites for 18h at various multiplicity of infection. After infection, HFFs were washed in PBS, seeded in round- bottom 96-well plates and incubated for 20 min at 4°C with fixable viability dye AF450 (eBioscience). Mouse anti-SAGl antibody was diluted in FACS buffer and incubated with HFF for 30 min at 4°C followed by extensive washing. HFF were fixed for 20 min with 4% paraformaldehyde in PBS at 4°C.
  • GRA6 coexists as a minor soluble form and a predominant transmembrane protein [Gendrin, 2010] that is inserted into membranes of the vacuole with a preferential IVN localization [Labruyere, 1999].
  • TgCEP.GFP.Luc shortened as TgCEP
  • HFlO-containing antigenic constructs coding either for the natural form of GRA6 (i.e. membrane-bound plus soluble) or for a fully soluble mutant (designated GRA6mb+sol and GRA6sol respectively, data not shown).
  • IVN tubular structures were absent from vacuoles containing GRA2-deficient parasites and only proteinaceous granular material was detected (data not shown).
  • we observed similar findings in infected BMM (data not shown).
  • Our results indicate that the IVN is generated in a GRA2-dependent manner not only in fibroblasts but also in professional antigen-presenting cells (APC) such as BMDC and BMM. They establish a model to study the role of IVN membranes on MHC I presentation by infected APC. Highly curved IVN membranes inhibit MHC I presentation of membrane-bound
  • gondii antigens in in vitro -infected APC yet do not impact presentation of a soluble antigen in a major way.
  • Highly curved IVN membranes is detrimental for priming of CD8 T cells specific for membrane-bound T. gondii antigen in vivo
  • TgRH.AGRA2 elicited a significantly higher proportion of HFlO-reactive CD8 T cells in both tissues examined, as compared to GRA2-expressing T. gondii, while no statistically significant difference was observed in the magnitude of the Tgd057-specific response.
  • Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole.
  • a helical membrane-binding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic 10, 1458-1470.
  • Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc Natl Acad Sci U S A 105, 13574-13579.

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Abstract

La présente invention concerne un mutant atténué de Toxoplasma gondii destiné à être utilisé dans la prévention ou le traitement d'un cancer ou de maladies infectieuses chez un sujet qui en a besoin, le mutant atténué 1) exprimant une ou plusieurs protéines GRA6 chimériques comportant un antigène à leur extrémité C et 2) ayant une mutation d'inactivation ou de perte de fonction du gène codant pour la protéine GRA2.
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WO2018002938A1 (fr) * 2016-06-29 2018-01-04 Ramot At Tel-Aviv University Ltd. Modification de parasites pour l'administration de protéines dans le système nerveux central.
KR20190021339A (ko) * 2016-06-29 2019-03-05 라모트 앳 텔-아비브 유니버시티 리미티드 중추신경계(cns)에 단백질을 전달하기 위한 조작된 기생충
CN109757100A (zh) * 2016-06-29 2019-05-14 特拉维夫大学拉莫特有限公司 用于向中枢神经系统（cns）递送蛋白质的工程改造的寄生虫
JP2019525744A (ja) * 2016-06-29 2019-09-12 ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッドＲａｍｏｔ Ａｔ Ｔｅｌ Ａｖｉｖ Ｕｎｉｖｅｒｓｉｔｙ Ｌｔｄ． タンパク質を中枢神経系（ｃｎｓ）に送達するための、組換え寄生虫
US11260081B2 (en) 2016-06-29 2022-03-01 Ramot At Tel-Aviv University Ltd. Engineered parasites for delivering protein to the central nervous system (CNS)
JP7148415B2 (ja) 2016-06-29 2022-10-05 ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッド タンパク質を中枢神経系（ｃｎｓ）に送達するための、組換え寄生虫
KR102513120B1 (ko) * 2016-06-29 2023-03-27 라모트 앳 텔-아비브 유니버시티 리미티드 중추신경계(cns)에 단백질을 전달하기 위한 조작된 기생충
JP7497397B2 (ja) 2016-06-29 2024-06-10 ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッド タンパク質を中枢神経系（ｃｎｓ）に送達するための、組換え寄生虫
CN111132683A (zh) * 2017-06-09 2020-05-08 图尔大学 用于治疗癌症和感染性疾病的新孢子虫
CN114164114A (zh) * 2021-12-08 2022-03-11 华南农业大学 弓形虫核酮糖-5-磷酸异构酶TgRPI基因编辑虫株及其应用

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