WO2016046321A1 - Method for treating cancer and infectious diseases - Google Patents

Abstract

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.

Description

METHOD FOR TREATING CANCER AND INFECTIOUS DISEASES

FIELD OF THE INVENTION:

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.

BACKGROUND OF THE INVENTION:

Recent work highlighted the value of using attenuated intracellular parasites as possible weapons in the arsenal of anti-cancer immunotherapy [Fox B. et al., 2013a and Fox B. el al, 2013b]. As strong inducers of 'Thl '-oriented immune responses, these parasites act as powerful adjuvants and may also serve as vaccine vectors for potentiating a specific T cell response against tumor antigens (Ags). The beneficial adjuvant effect of intracellular parasite infection has been reported in several experimental models [Darani HY et al., 2012]. For instance, 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. et al., 2013b]. In the latter case, presence of T. gondii in the tumor environment caused maturation and conversion of DC into immunostimulatory cells. These inhibitory effects on tumors are reminiscent of the pioneering idea proposed in 1893 that stimulation of the immune system by microorganisms could be exploited to combat cancer [Coley WB et al, 1991].

Beyond their role as adjuvant, 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. 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]. Whereas the rhoptry content is injected directly into the host cytosol upon initial contact, dense granule material is thought to be secreted after invasion has completed, and then to build up in the mature vacuole [Ravindran, 2008]. A prominent feature of the vacuole is the presence of an IntraVacuolar Network (IVN) of highly curved membraneous tubules that connect parasites together and to the vacuole limiting membrane [Cesbron-Delauw MF et al, 2008]. This network has also been coined Membranous Nanotubular Network [Travier L. et al., 2008]. We previously revealed the implication of two dense granule-secreted proteins (GRA2 [Travier L. et al., 2008] and GRA6 [Lecordier L et al, 1995] in IVN biogenesis. Shortly following release in the vacuolar space, GRA2 relocalizes to the posterior end of the parasite and associates with membrane whorls and vesicles [Sibley LD et al., 1995]. Deletion of the GRA2 gene and complementation with truncated versions of GRA2 indicated that both its N-terminal domain and its 3 central AAH are necessary for IVN formation [Mercier C. et al., 2002 and Travier L et al, 2008]. Instead, disruption of the GRA6 gene led to the IVN replacement with smaller tubules and vesicles, suggesting that GRA6 is not the main effector but that it may help stabilize the membranous tubules initiated by GRA2 [Mercier C et al, 2002]. The IVN was initially proposed to promote ordered arrangement and synchronous division of the dividing parasites [Magno RC et al, 2005 and Travier L et al, 2008] but this idea has lacked formal demonstration. 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. 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. gondii, the fact that processing is dependent on proteasome activity and TAP transport [Bertholet S et al, 2006; Gubbels MJ et al, 2005; Blanchard N et al, 2008 and Grover HS et al, 2014] suggests that parasite antigenic precursors have to transit through the cytosol. Passage of T. gondii antigens to the cytosol may be facilitated by the recruitment of components of the host ER-associated degradation machinery onto the vacuole membrane [Cebrian I et al, 2011 and Goldszmid RS et al, 2009]. However the details of this export pathway remain elusive and its regulation has not been elucidated.

Strategies of using attenuated Toxoplasma gondii mutants as vaccines in the prevention or treatment of cancer already exist [see for example Baird JR. et al 2013a; Baird JR. et al 2013b; Fox BA et al 2013a; Fox BA et al 2013b and patent applications US 8282942, US 20120045477 or WO 2013059411]. These strategies have consisted in injecting an attenuated strain of T. gondii intra-tumorally and exploiting the adjuvant effect of T. gondii that activates innate immune cells (dendritic cells, Natural Killer cells) and in turn promotes tumor-specific CD8 T cells. However, these strategies have failed to define an optimal, most immunogenic configuration for expression 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. SUMMARY OF THE INVENTION:

By examining to which extent membranes at the host-parasite interface (IVN and vacuole limiting membrane) regulate the immunogenicity of T. gondii proteins and by using reverse genetics in T. gondii and in vitro and in vivo antigen presentation measurements, the inventors show 1/ that binding of GRA6 protein to the limiting membrane of the vacuole is essential for efficient MHC class I presentation and 21 that disruption of the GRA2 gene, through removal of the IVN, enhances this presentation.

Thus, 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. DETAILED DESCRIPTION OF THE INVENTION:

Attenuated mutant of Toxoplasma gondii and uses thereof

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.

According to the invention, the attenuated mutant of Toxoplasma gondii may be used for the prevention or the treatment of cancer or of infectious diseases.

Thus, 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.

As used herein, the term "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.

As used herein, the term "GRA2" 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. As used herein, 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. In one embodiment, 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. In all cases 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.

Thus, according to the invention, the attenuated mutant of Toxoplasma gondii may express one or more than one chimeric GRA6 proteins with an antigen at its C-terminus. In other words, 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.

Thus, in one embodiment, 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.

In one embodiment, 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.

As used herein, the terms "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. According to another embodiment, 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. In a further aspect, 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.

Accordingly, 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. For example, 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. 123(1): 1-10, or by using the CRISPR/Cas9-based genome editing method (Shen B et al Mbio 2014; Sidik SM et al PloS One 2014). In general, 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. Upon selection, i.e., marker protein expression or genomic DNA analysis, a knockout mutant is obtained. In particular embodiments, 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. Disruption of all or a portion of a gene of interest (like GRA2) 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. As is known to the skilled artisan, subsequent restriction endonuclease digestion and Southern blot analysis of the mutant T. gondii genomic DNA can be used to confirm the knockout.

As will be appreciated by the skilled artisan, 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.

In a particular embodiment, the attenuated mutant T. gondii of the invention is a γ- irradiated attenuated mutant strain of T. gondii. The use of γ 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]. Specifically, 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. In particular, when attenuated by γ irradiation, it is desirable that the attenuated mutant maintain the ability to invade cells, including DC and myeloid cells, to provide optimal antitumor or anti-infectious responses.

As used herein the term "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).

In one embodiment, the antigen according to the invention is an antigenic peptide.

In a particular embodiment, the antigen may be a "Tumor antigen". As used herein, the term "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.

In one embodiment, 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.

Accordingly, in one embodiment of the present invention, 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. In this regard, it is desirable that the promoter employed is recognizable by T. gondii. Moreover, it is desirable that the promoter promotes transcription of the protein coding sequence when the T. gondii is inside mammalian cells. To this end, particular embodiments embrace the use of a T. gondii promoter. Known promoter and other regulatory elements (e.g., 5' UTR, 3' UTR, etc.) 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). Alternatively, 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. Moreover, it is contemplated that 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. As used herein, the term "attenuated" refers to a weakened and/or less vigorous strain of T. gondii. Desirably, 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 γ-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). Specifically, 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., NT2 or NT3). Indeed, any single knockout or combination of knockouts is contemplated to achieve an attenuated strain. By way of illustration, 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. 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. According to the invention, 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). In another embodiment, 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. This could include 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. Such 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. In one embodiment, the cancer may be any solid cancer. Typically, 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. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), 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. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non- Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), 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. 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).

In one embodiment, infectious diseases may be a disease caused by a virus, a bacterium, a parasite or a fungus.

In a particular embodiment, 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.

In a particular embodiment, 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.

In a particular embodiment, infectious diseases may be caused by a parasite like Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania major or Leishmania donovani.

Thus, in one embodiment, 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 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.

Vaccine composition In a further object of the invention, the attenuated mutant of Toxoplasma gondii of the invention may be used for the preparation of a vaccine composition.

Hence, the present invention also provides a vaccine composition comprising the attenuated mutant of Toxoplasma gondii according to the invention.

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. Such 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.

As used herein, the term "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.

As used herein, the term "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. Non-limiting examples of some commonly used vaccine adjuvants include insoluble aluminum compounds, calcium phosphate, liposomes, Virosomes™, 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.

Examples of adjuvants that may be effective 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. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, 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, γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). 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 Mycobacterium tuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxide adjuvant.

As used herein, the term 'TLR4 agonist" denotes a compound or a molecule which bind the Toll-like receptor 4 and active it. According to the invention, 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.

In a particular embodiment, the TLR4 agonist according to the invention is selected from the group consisting of the LPS or monophosphoryl lipid A (MPL).

Various 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. For example, 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. 71(5):2498-507; Ogawa T. et al., 2002. Int Immunol. 14(11): 1325-32). 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 (DPLA) 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. PCT/US2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865; PCT/US 2006/042051. 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). Other examples include those described in Orr MT, Duthie MS, Windish HP, Lucas EA, Guderian JA, Hudson TE, Shaverdian N, O'Donnell J, Desbien AL, Reed SG, Coler RN. MyD88 and TRIF synergistic interaction is required for THl-cell polarization with a synthetic TLR4 agonist adjuvant. Eur J Immunol. 2013 May 29. doi: 10.1002/eji.201243124.; Lambert SL, Yang CF, Liu Z, Sweetwood R, Zhao J, Cheng L, Jin H, Woo J. Molecular and cellular response profiles induced by the TLR4 agonist-based adjuvant Glucopyranosyl Lipid A. PLoS One. 2012;7(12):e51618. doi: 10.1371/journal.pone.0051618. Epub 2012 Dec 28.

As used herein, the term 'TLR9 agonist" denotes a compound or a molecule that binds the Toll-like receptor 9 and actives it. According to the invention, a TLR9 agonist may be selected from the group consisting of CpG oligonucleotides (ODN) and its derivatives.

In particular embodiment, the TLR9 agonist is the CpG (ODN).

Examples of TLR9 agonists (include nucleic acids comprising the sequence 5 -CG-3' (a

"CpG nucleic acid") in certain aspects C is unmethylated. The terms "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. Thus 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. In some embodiments, 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. Examples of non- limiting TLR9 agonist-enriched plasmids are described in Roman et al. (1997). In general, a TLR9 agonist used in a subject composition comprises at least one unmethylated CpG motif. In some embodiments, 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. In other embodiments, 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. In some of these embodiments, the at least two additional CG dinucleotides are spaced three nucleotides, two nucleotides, or one nucleotide apart. In some of these embodiments, the at least two additional CG dinucleotides are contiguous with one another. In some of these embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, at the 5' end of the nucleic acid. In other embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, and where the (TCG)n sequence is flanked by one nucleotide, two nucleotides, three nucleotides, four nucleotides, or five nucleotides, on the 5' end of the (TCG)n sequence. 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.

Examples of others 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. For example, 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.

Therefore, 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.

Those skilled in the art will be able to select an antigen appropriate for treating a particular pathological condition and will know how to determine whether an isolated antigen is favored in a particular vaccine formulation.

In another particular embodiment, 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.

Examples of 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.

Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ- Amine or NZ-Amine AS. Examples of 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.

Conventional 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. In addition to the active ingredient(s), 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.

Besides inert diluents, the oral compositions can also include 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.

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). 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.

Examples of suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention 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. 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.

These 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. In addition, 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. 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. While 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 μg to about 500 μg (and in particular 50 μg to about 500 μg) of the adjuvant compound of the invention per dose.

Although 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.

For example, 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.

For example, 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.

Those skilled in the art will know how to determine for a particular vaccine composition, the appropriate antigen payload, route of immunization, volume of dose, purity of antigen, and vaccination regimen useful to treat a particular pathological condition in a particular animal species.

In a vaccination protocol, 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.

As a general rule, 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. The route of administration contemplated by the present invention will depend upon the antigen.

According to the invention, the vaccinal composition of the present invention may be used in human health or animal health. In other word, 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:

Figure 1: Association of T. gondii dominant antigen to lipid membranes of the vacuole promotes MHC I presentation by infected cells

HFIO presentation by BMM infected in vitro for 24h with the indicated TgCEP, assessed with the CTgEZ.4 CD8 hybridoma. Representative of 3 independent experiments.

Figure 2: In infected bone marrow-derived macrophages (BMM), highly curved membranes of the IVN decrease MHC I presentation of membrane-bound antigen

L^d-HF10 presentation by BMM infected for 24h with the indicated parasites, assessed with the CTgEZ.4 hybridoma. Data depicted on all panels are representative of at least 3 independent experiments. Figure 3: Immunization with mutant T. gondii devoid of IVN enhances T cell response to GRA6 antigen but does not affect T cell response to Tgd057 antigen

(A,B) Ex vivo IFNy intracellular staining of splenocytes (A) or peritoneal exudate cells (B) restimulated with the HF10 (GRA6n) or SVL8 (Tgd057) peptide. Bars represent the mean +/- SEM. *: P < 0.05 ; **: P < 0.005 ; ***: P < 0.005. Data pooled from 2 independent experiments.

Figure 4: Disruption of highly curved IVN enhances presence of T. gondii GRA6 antigen at the vacuole limiting membrane

Ratio of HF10 fluorescence signal at vacuole limiting membrane (ROIl - ROI2) over total HF10 fluorescence in the whole vacuole (ROIl). Each dot represents the median value of 3 ratio measurements performed on 3 middle sections of a single vacuole. P < 0.05

EXAMPLES: Material & Methods

Ethics statement

Animal studies were carried out under the control of the National Veterinary Services and in accordance with European regulations (EEC directive 86/609 dated 24 November 1986). The protocol was approved by the Regional Ethics Committee of Midi-Pyrenees Region (Approval MP/01/29/09/10). Mice, parasites and antibodies

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-γ (XMG1.2) (eBioscience). Primary antibodies for Western blot and immunofluorescence were rabbit anti- Tgd057 serum and purified rabbit anti-HFlO serum (custom-made, Biotem, Grenoble), rabbit anti-OVA (Sigma), mouse anti-GRA2 (17-179, Biotem), mouse anti-GRAl (gift from M.F. Delauw, Grenoble), mouse anti-GRA3 (gift from M.F. Delauw, Grenoble), mouse anti-SAGl (TP3, Santa Cruz).

Plasmid constructs and parasite transfection

Parasite transfections were performed as previously described (Feliu et al, 2013). Briefly, 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.

Western blot and subcellular fractionation

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).

BMM and BMDC differentiation and antigen presentation assays

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. Sedlik (Curie Institute, Paris, France) respectively. When non- fluorescent, the tachyzoites were labeled with CMTMR. B6D2 Fl BMM and BMDC were seeded into flat-bottom 96-well plates and infected for 8h or 24 h with tachyzoites with serially diluted tachyzoites. In all experiments, the proportion of infected (GFP⁺, YFP⁺ or CMTMR⁺) cells was controlled by flow cytometry 6 h post-infection. Presentation of the GRA6(II)-derived HF10 by L^d, GRA4-derived SM9 by L^d, OVA-derived SL8 by K^b and 28m-derived AS 15 by IA^b were assessed by 16 h incubation with the CTgEZ.4 (Blanchard et al, 2008), BDSM9Z (Feliu et al, 2013), B3Z (Cebrian et al, 2011) and BTgOlZ (Graver et al., 2012) reporter hybridomas respectively. TCR-mediated stimulation of β- galactosidase production by the hybridomas was quantified using a chromogenic substrate: chlorophenol red-P-D-galactopyranoside (CPRG, Roche). Cleavage of the CPRG by β- galactosidase releases a purple product, which absorbance was read at 595 nm with a reference at 655 nm. Ex vivo analysis of T cell response

Mice were immunized twice 3 weeks apart with 10⁶ tachyzoites that were previously γ- irradiated (120 Gy) and filtered through a 3 μιη filter (Millipore). Mice were euthanized 4 days after the second immunization. Peritoneal exudate cells (PEC) were recovered by lavage with 10 ml cold PBS. 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 μιη EDTA, 160 mM NH4C1 and 10 mM NaHC03). Production of IFN-γ following restimulation with 100 nM of the indicated synthetic peptides was assessed as described previously (Feliu et al, 2013), following the manufacturer's instructions (Cytofix/Cytopermkit, BD Pharmingen). Samples were run on a FC500 (Beckman- Coulter) or Fortessa (Becton Dickinson) flow cytometer and analyzed using Flow Jo software.

Immunofluorescence

Monolayer of HFF grown on Lab-Tek II chamber slides CC2 (Thermo Scientific Nunc) 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. Cells were labeled with secondary goat anti-rabbit IgG AF555 and anti-mouse IgG AF633 (Invitrogen) for 20 min at RT. Coverslips were mounted on Lab- Tek slides with Vectashield medium with DAPI (Vector Labs). Z-stacks were acquired with a 63X objective on a Zeiss LSM710 confocal microscope and analyzed using Image J software. For more details on the quantifications of limiting membrane GRA6 over total GRA6, see Supplemental Experimental Procedures .

Statistical analysis

Prism software (GraphPad) was used for statistical analyses. All P values were calculated with the two-tailed Mann- Whitney test (nonparametric).

Plasmid constructs

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.

Name Purpose Host strain

Introduce GRA6(II) with ble

pBTH/ GRA6(II)/BLE TgRH AGRA6(I) selection

TgRH AGRA6(I) pTko .mCherry/ GRA2/HPT/ GRA2 Delete GRA2(I) with hpt selection

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 (Donald and Roos, 1993) for dhfr

selection

pGRA2/Ble/GRA2 (Mercier et al, TgRH YFP

Delete GRA2(I) with ble selection

1998b) SAG 1 -OVA pBLUESCRIPT/GRA6(I)Nter- Introduce GRA6mb, cotransfected

TgCEP GFP Luc TM-Cter-HF10 with pMiniHPT for hpt selection

pBLUESCRIPT/SAGlNter- Introduce GRA6sol, cotransfected

TgCEP GFP Luc GRA6(I)TM-Cter-HF 10 with pMiniHPT for hpt selection

Introduce OVAsol, cotransfected

pBLUESCRIPT/SAGlNter-OVA TgRH ΔΗΡΤ

with pMiniHPT for hpt selection

pBLUESCRIPT/GRA6(I)Nter- Introduce OVAsol, cotransfected

TgRH ΔΗΡΤ TM-OVA with pMiniHPT for hpt selection

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.

All of the newly constructs designed to be expressed in the parasite were cloned under the control of the regulatory regions of endogenous GRA5 (promoter, 5' and 3' untranslated regions) because the GRA5 promoter allows high-level expression of fusion proteins (Mercier et al, 1993).

The constructs were cloned into pBLUESCRIPT vector using the In-Fusion HD Cloning kit (Clontech).

Specific fragments of the open reading frame (ORF) of GRA6, GRA5, SAG1 or OVA were then amplified with primers containing 25-50 bases complementary to the DNA fragment to be amplified and tailed with 15 bases complementary to the adjacent DNA fragments in the final construct. The final products were cloned into the Smal and Bglll sites of the pBLUESCRIPT plasmid in frame with the GRA5 5' and the 3' UTR and to remove the HF tag.

Generation of AGRA2 mutants

To disrupt the GRA2 gene in TgRH.AGRA6(I).GRA6(II)tg, the plasmid pTko.mCherry.GRA2 targeting, provided by J. Boothroyd, was used. Freshly egressed tachyzoites were transfected by electroporation using 10⁷ cells combined with 50 μg of linearized plasmid, selected with xanthine and mycophenolic acid and cloned by limiting dilution in flat-bottom 96-well plates. Knock-out clones were complemented by cotransfection of 10⁷ cells with 50 μg of the linearized plasmid pCG2-5'PstI-3'SalI (Mercier et al, 1993) mixed with 5 μg of the linearized plasmid pDHFR-TSc3 (Donald and Roos, 1993) and selected for pyrimethamine.

To disrupt the GRA2 gene in TgRH.YFP.OVA, 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.

Generation of transgenic parasites

To address HF10 epitope at diverse locations inside parasitophorous vacuole, 2 or 3 plasmids of chimeric GRA6 were used. 2xl0⁷ CEP.Ahpt.GFP.Luc (Kamau et al., 2011) were cotransfected by electroporation with 50 μg of the linearized plasmid of interest mixed with 5 μg of the linearized plasmid pminiHXGPRT (Donald et al., 1996). After a selection with xanthine and mycophenolic acid, stable clones were cloned by limiting dilution in 96-well microtiter plates.

To mediate OVA at various locations in the PV, 50 μg of the linearized ptubP30-OVA sagCAT (Gubbels et al, 2005), pGRA5-OVA or pGRA6-OVA with 5 μg of the linearized pTUB/CAT/SAGl (Kim et al, 1993) were electroporated into TgRH.Ahpt (Donald et al, 1996).

Chloramphenicol was added to allow selection and stable clones were isolated by limiting dilution. Oligonucleotides

Sequences of all primers used in this study for cloning and validation of parasite transfectants are available upon request. They were designed based on NCBI and sequences of type I (GT1), II (Me49) or III (VEG) parasites publicly available on www.ToxoDB.org. GRA6: TGGT1 275440, GRA2: TGGT1 227620, GRA5 : TGGT1 286450, SAG1 (P30/SRS29B): TGGT1 233460, chicken ovalbumin (OVA): AY223553.1

Parasite transfection

For parasite transfections, 1.5xl0⁷ tachyzoites were electroporated with 50 μg of

Hindlll-linearized plasmid DNA and inoculated in 4 confluent HFF flasks in order to obtain up to 4 independent clones. The next day, 25 μ§/ι 1 mycophenolic acid and 50 μ§/ι 1 xanthine were added for selection. After 2 passages, resistant tachyzoites were cloned by limiting dilution and presence of the transgene was verified by PCR. For each construct, one clone that acquired resistance but no transgene was kept as HXGPRT⁺ control.

Secretion assay

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. Cells were incubated with AF647-coupled rabbit anti-HFl O or anti-HY10 antibodies and AF555-coupled anti-mouse IgG secondary Abs (Invitrogen) diluted in PBS, 0.2% BSA, 0.05%> saponin for lh at 4°C. Samples were washed, run on Fortessa flow cytometer (BD Biosciences) and analyzed with Flow Jo software.

Results Membrane association of T. gondii antigen is beneficial for access to the MHC I pathway

We first chose to determine whether membrane binding affects MHC I antigen processing by parasite-infected cells. To this end, we set out to perturb trafficking of a T. gondii- secreted membrane-bound antigen. We focused on GRA6 and its naturally processed decamer epitope (HPGSVNEFDF, HF10, SEQ ID NO: l), which is presented by L^d MHC I and elicits dominant and protective CD8 responses in T. gondii-mfQcted mice [Blanchard, 2008 and Feliu, 2013]. Following release in the vacuole, 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]. We first engineered an avirulent type III "HF10 epitope-null" parasite (TgCEP.GFP.Luc, shortened as TgCEP) [Feliu, 2013] to stably express 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). Design of the fully soluble GRA6sol mutant was based on our previous observation that the N-terminal domain of GRA6 is responsible for its selective membrane association [Gendrin, 2010]. Hence, we replaced GRA6 N-terminal domain with that of another T. gondii protein: SAG1/P30. By immunofluorescence we then verified that GRA6mb+sol and GRA6sol antigenic constructs were properly released in the vacuole by transgenic TgCEP. Using prototypical markers of the vacuolar lumen (GRA1) and the vacuole limiting membrane (GRA3), we observed that GRA6mb+sol was present both at the vacuole limiting membrane as well as in the lumen (most likely bound to the IVN, but which cannot be resolved by light microscopy), whereas GRA6sol displayed only limited overlap with the limiting membrane GRA3 marker (data not shown). When used to infect bone marrow-derived macrophages (BMM), TgCEP. GRA6mb+sol and TgCEP. GRA6sol transgenic parasites led to similar infection rates (data not shown) but HF 10 presentation was almost completely abrogated with the soluble version (Figure 1). This difference could not be explained by a lower expression level of GRA6sol within the tachyzoites (data not shown). These data suggest that disrupting lipid association (i.e. enhancing solubility) of a membrane-bound antigen is detrimental for efficient processing and MHC I presentation by T. gondii-mfQcted cells.

GRA2-dependent IVN biogenesis in dendritic cells and macrophages

Knowing that membrane association positively regulates MHC I presentation of membrane-bound antigens, we next investigated how highly curved membranes of the IVN may specifically impact this process. Because the GRA2 protein is a major effector of IVN biogenesis [Mercier, 2002], we decided to delete the GRA2 gene and assess the consequence on presentation of the GRA6-derived epitope. As gene deletion is knowingly difficult to achieve in avirulent type II or III parasites, we selected the more genetically tractable type I TgRH to disrupt GRA2 by double homologous recombination, after stably expressing an HF10- containing GRA6. We also generated a control parasite where GRA2 was added back. These parasites are later designated as TgRH, TgRH.AGRA2 and TgRH.AGRA2.+GRA2 (data not shown). So far, the IVN and its disruption in GRA2-deficient parasites has been reported in fibroblasts but has never been assessed in immune cells. To ensure that our mutants have the expected phenotype, we examined the presence and shape of the IVN in bone marrow-derived dendritic cells (BMDC) by TEM (data not shown). Membrane-bound tubulo vesicular material was observed in the vacuole lumen around the parasites in BMDC infected with TgRH or the complemented TgRH.AGRA2.+GRA2 (data not shown). As expected, 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

T. gondii antigen in vitro

To interrogate the role of IVN in MHC I presentation of the membrane-bound GRA6 antigen, we infected BMM in vitro with our 3 new parasites. All 3 parasites led to similar infection rates whether or not they expressed GRA2 (data not shown) but HF10 presentation was largely enhanced when GRA2 was deleted (Figure 2). This was neither related to a difference in GRA6 expression level by the transgenic parasites (data not shown) nor to a global increase of MHC I surface levels with TgRH AGRA2, since presentation of an exogenously pulsed SM9 peptide by L^d on infected BMM was similar between all conditions (data not shown). As GRA2 and GRA6 may directly or indirectly interact, absence of GRA2 could interfere with (more precisely, promote) secretion of GRA6 in the vacuolar space. To quantify the amount of GRA6 released in the vacuole, we designed a flow cytometry-based assay relying on the fact that saponin permeabilizes the host cell plasma membrane plus the vacuole limiting membrane, but not the parasite itself when intracellular (data not shown). By gating out cells with attached extracellular tachyzoites (= SAG 1 -positive) and by detecting GRA6 with an HFlO-specific antibody, we found that the amount of vacuolar GRA6 was independent from the presence of GRA2 (data not shown). We then questioned whether increase in MHC I presentation in the absence of GRA2 would apply to all T. gondii-denved antigens or would be restricted to membrane-bound antigens. We deleted GRA2 in TgRH.YFP parasites expressing the OVAsol antigen [Gubbels, 2005] and observed no major consequence on OVA-derived SL8 presentation by Kb MHC I (data not shown). Importantly, we made identical observations in BMDC (data not shown), which are the most potent cell subset for priming parasite-specific CD 8 T cells in vivo. Our data show that highly curved membranes of the IVN negatively regulate MHC I presentation of membrane-bound T. 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

To strengthen the relevance of our findings, we then investigated the influence of the IVN on the induction of CD8 T cell responses in vivo using a protocol of prime/boost immunization with irradiated TgRH transfectants expressing GRA2 or not (data not shown). Four days following the second immunization, we assessed the magnitude of CD8 T cell responses to the dominant GRA6-derived HF10 peptide and the subdominant Tgd057-derived SVL8 peptide (Wilson et al, 2010) in the spleen (Figure 3A) and the peritoneum (Figure 3B). 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. Taken together, our in vitro and in vivo data demonstrate that highly curved IVN membranes inhibit MHC I presentation in vitro and dampens CD8 T cell priming to a membrane-bound dominant T. gondii antigen.

Disruption of IVN increases its targeting at the vacuole limiting membrane

Hitherto our data indicate that while membrane insertion of T. gondii antigens is beneficial for their MHC I presentation, targeting to the IVN is detrimental. To understand this seemingly paradoxical result, we examined the localization of GRA6 in the presence or absence of GRA2/IVN. We quantified the fluorescence signal of GRA6 (anti-HFlO antibody) at the vacuole surrounding membrane versus the entire vacuole in a panel of infected fibroblasts by confocal microscopy (data not shown). Absence of GRA2 resulted in a higher ratio of limiting membrane over total fluorescence (Figure 4). Therefore, our data are compatible with the following working model (data not shown): presence at the vacuole limiting membrane favorably regulates access to the MHC I pathway but the IVN downregulates MHC I presentation of membrane-bound GRA6 by 'sequestering' it away from the limiting membrane.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Baird Jason R., Barbara A. Fox, Kiah L. Sanders, et al. Avirulent Toxoplasma gondii Generates Therapeutic Antitumor Immunity by Reversing Immunosuppression in the Ovarian Cancer Microenvironment. Cancer Res 2013a;73:3842-3851.

Baird Jason R., Katelyn T. Byrne, Patrick H. Lizotte, Seiko Fiering Bosenberg, David W. Mullins, Mary Jo Turk and Steven Mee Rie Sheen, Barbara A. Fox, David J. Bzik, Marcus. Immune-Mediated Regression of Established B16F10 Melanoma by Intratumoral Injection of Attenuated Toxoplasma gondii Protects against Rechallenge. J Immunol 2013b; 190:469-478.

Bertholet, S., Goldszmid, R., Morrot, A., Debrabant, A., Afrin, F., Collazo-Custodio, C, Houde, M., Desjardins, M., Sher, A., and Sacks, D. (2006). Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo. J Immunol 177, 3525-3533.

Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J., Robey, E.A., and Shastri, N. (2008). Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Nat Immunol 9, 937-944.

Blum, J.S., Wearsch, P.A., and Cresswell, P. (2013). Pathways of antigen processing. Annu Rev Immunol 31, 443-473.

Cebrian, I., Visentin, G., Blanchard, N., Jouve, M., Bobard, A., Moita, C, Enninga, J., Moita, L.F., Amigorena, S., and Savina, A. (2011). Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell 147, 1355-1368.

Cerede Odile, Dubremetz J.F., Soete M., Deslee D., Vial H., Bout D., Lebrun M., Synergistic role of micronemal proteins in Toxoplasma gondii virulence, J. Exp. Med. (2005) 201 :453-463.

Cesbron-Delauw, M.F., Gendrin, C, Travier, L., Ruffiot, P., and Mercier, C. (2008). Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole. Traffic 9, 657- 664.

Chen, L., He, Z., Qin, L., Li, Q., Shi, X., Zhao, S., Zhong, N., and Chen, X. (2011). Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity. PloS one 6, e24407.

Coley, W.B. (1991). The treatment of malignant tumors by repeated inoculations of erysipelas. With a report of ten original cases. 1893. Clin Orthop Relat Res, 3-11.

Darani, H.Y., and Yousefi, M. (2012). Parasites and cancers: parasite antigens as possible targets for cancer immunotherapy. Future Oncol 8, 1529-1535. Donald, R.G., Carter, D., Ullman, B., and Roos, D.S. (1996). Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. The Journal of biological chemistry 271, 14010-14019.

Donald, R.G., and Roos, D.S. (1993). Stable molecular transformation of Toxoplasma gondii: a selectable dihydro folate reductase-thymidylate synthase marker based on drug- resistance mutations in malaria. Proceedings of the National Academy of Sciences of the United States of America 90, 11703-11707.

Dou, Z., McGovern, O.L., Di Cristina, M., and Carruthers, V.B. (2014). Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins. MBio 5.

Dubey JP1, Thayer DW, Speer CA, Shen SK. Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts. Int J Parasitol. 1998 Mar;28(3):369- 75.

Feliu, V., Vasseur, V., Grover, H.S., Chu, H.H., Brown, M.J., Wang, J., Boyle, J.P., Robey, E.A., Shastri, N., and Blanchard, N. (2013). Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite. PLoS pathogens 9, el 003449

Fox Barbara A, Kiah L Sanders, and David J Bzik. Non-replicating Toxoplasma gondii reverses tumor-associated immunosuppression. Oncolmmunology 2:11, e26296; 2013a.

Fox Barbara A., Kiah L. Sanders, Shan Chen, and David J. Bzik. Targeting tumors with nonreplicating Toxoplasma gondii uracil auxotroph vaccines. Trends in Parasitology, September 2013b.

Gendrin, C, Bittame, A., Mercier, C, and Cesbron-Delauw, M.F. (2010). Post- translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasite- containing vacuole is driven by its N-terminal domain. Int J Parasitol 40, 1325-1334.

Goldszmid, R.S., Coppens, I., Lev, A., Caspar, P., Mellman, I., and Sher, A. (2009). Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross- presentation in Toxoplasma gondii- fectGd dendritic cells. J Exp Med 206, 399-410.

Grover, H.S., Chu, H.H., Kelly, F.D., Yang, S.J., Reese, M.L., Blanchard, N., Gonzalez, F., Chan, S.W., Boothroyd, J.C., Shastri, N., et al. (2014). Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses. Cell reports.

Gubbels, M.J., Striepen, B., Shastri, N., Turkoz, M., and Robey, E.A. (2005). Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect Immun 73, 703-711. Hiramoto RM1, Galisteo AJ, do Nascimento N, de Andrade HF Jr. 200 Gy sterilised Toxoplasma gondii tachyzoites maintain metabolic functions and mammalian cell invasion, eliciting cellular immunity and cytokine response similar to natural infection in mice. Vaccine. 2002 May 15;20(16):2072-81.

Kook Jl, Oh SH, Yun CK, Chai JY. Effects of gamma- irradiation on intracellular proliferation of Toxoplasma gondii RH tachyzoites. Korean J Parasitol. 1995 Sep;33(3): 173- 8.Labruyere, E., Lingnau, M., Mercier, C, and Sibley, L.D. (1999). Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii. Mol Biochem Parasitol 102, 311-324.

Lecordier, L., Moleon-Borodowsky, I., Dubremetz, J.F., Tourvieille, B., Mercier, C,

Deslee, D., Capron, A., and Cesbron-Delauw, M.F. (1995). Characterization of a dense granule antigen of Toxoplasma gondii (GRA6) associated to the network of the parasitophorous vacuole. Mol Biochem Parasitol 70, 85-94.

Magno, R.C., Lemgruber, L., Vommaro, R.C., De Souza, W., and Attias, M. (2005). Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole. Microsc Res Tech 67, 45-52.

Marie-Noelle Mevelec, Celine Ducournau, Alaa Bassuny Ismael, Michel Olivier, Edouard Seche, Maryse Lebrun, Daniel Bout, Isabelle Dimier Poisson (2010). Micl-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep. Vet. Res. (2010) 41 :49

Mercier, C, Lecordier, L., Darcy, F., Deslee, D., Murray, A., Tourvieille, B., Maes, P., Capron, A., and Cesbron-Delauw, M.F. (1993). Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii. Mol Biochem Parasitol 58, 71-82.

Mercier, C, Howe, D.K., Mordue, D., Lingnau, M., and Sibley, L.D. (1998). Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice. Infect Immun 66, 4176-4182.

Mercier, C, Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., and Cesbron- Delauw, M.F. (2002). Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol Biol Cell 13, 2397-2409.

Niedelman, W., Gold, D.A., Rosowski, E.E., Sprokholt, J.K, Lim, D., Farid Arenas, A., Melo, M.B., Spooner, E., Yaffe, M.B., and Saeij, J.P. (2012). The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response. PLoS pathogens 8, el002784. Ravindran, S., and Boothroyd, J.C. (2008). Secretion of proteins into host cells by Apicomplexan parasites. Traffic 9, 647-656.

Reese, M.L., and Boothroyd, J.C. (2009). A helical membrane-binding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic 10, 1458-1470.

Salehi, M., Taheri, T., Mohit, E., Zahedifard, F., Seyed, N., Taslimi, Y., Sattari, M.,

Bolhassani, A., and Rafati, S. (2012). Recombinant Leishmania tarentolae encoding the HPV type 16 E7 gene in tumor mice model. Immunotherapy 4, 1107-1120.

Shen B, Brown KM, Lee TD, Sibley LD. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. MBio. 2014 May 13;5(3):e01114-14. doi: 10.1128/mBio.Ol 114-14.

Sibley, L.D., Niesman, I.R., Parmley, S.F., and Cesbron-Delauw, M.F. (1995). Regulated secretion of multi- lamellar vesicles leads to formation of a tubulo -vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J Cell Sci 108 ( Pt 4), 1669-1677.

Sibley, L.D. (2011). Invasion and intracellular survival by protozoan parasites. Immunological reviews 240, 72-91.

Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One. 2014 Jun 27;9(6):el00450. doi: 10.1371/journal.pone.0100450. eCollection 2014.

Travier, L., Mondragon, R., Dubremetz, J.F., Musset, K., Mondragon, M., Gonzalez, S., Cesbron-Delauw, M.F., and Mercier, C. (2008). Functional domains of the Toxoplasma GRA2 protein in the formation of the membranous nanotubular network of the parasitophorous vacuole. Int J Parasitol 38, 757-773.

Van Dooren, G.G., Tomova, C, Agrawal, S., Humbel, B.M., and Striepen, B. (2008). Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc Natl Acad Sci U S A 105, 13574-13579.

Claims

CLAIMS:

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.

An attenuated mutant of Toxoplasma gondii for use in the prevention or 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 attenuated mutant of Toxoplasma gondii according to claims 1 or 2 for use in the prevention or treatment of cancer.

A vaccine composition comprising the attenuated mutant of Toxoplasma gondii according to claims 1 or 2.

The vaccine composition according to claim 4 which further comprises an adjuvant.

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.