NEOSPORA FOR USE IN TREATING CANCER AND INFECTIOUS DISEASES

Abstract

The present invention relates to a strain of Neospora caninum for use in treating cancer or infectious diseases.

Claims

1. A method for treating a chronic infectious disease in a subject, comprising administering to the subject at least one strain of Neospora caninum, wherein said chronic infectious disease is selected from chronic virus infection and chronic bacterial infection.

2. The method according to claim 1, wherein Neospora caninum is a wild type strain.

3. The method according to claim 1, wherein Neospora caninum is a mutant strain characterized by an over-expression of GRA15 protein, and/or by an under-expression of ROP16 protein.

4. The method according to claim 1, wherein the strain of Neospora caninum is at a tachyzoite stage.

5. The method according to claim 1, wherein said chronic infectious disease is associated with or induces an immunosuppression, and is selected from the group consisting of tuberculosis and HIV.

6. The method according to claim 1, wherein said at least one strain of Neospora caninum is comprised in a composition in association with an excipient.

7. The method according to claim 1, wherein said at least one strain of Neospora caninum is comprised in a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

8. The method according to claim 1, wherein said at least one strain of Neospora caninum is comprised in a vaccine composition.

9. The method according to claim 1, wherein said at least one strain of Neospora caninum is comprised in a vaccine composition comprising an adjuvant.

10. The method according to claim 1, wherein said at least one strain of Neospora caninum is administered to the subject via subcutaneous, intradermal or intratumoral routes.

11. The method according to claim 1, wherein the amount of cells of Neospora caninum administered to the subject is ranging from about 10.sup.4 to about 10.sup.11.

12. A mutant strain of Neospora caninum characterized by an over-expression of GRA15 protein, and/or an under-expression of ROP16 protein.

13. An ex vivo method for activating T cells, comprising contacting T cells with a strain of Neospora caninum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0149] FIG. 1 is a dose-response curve showing the percent survival of mice after the administration of different doses of wild type strains of Neospora caninum-1 (NC-1).

[0150] FIG. 2 is a histogram showing the seric IFNgamma production at day 2 or day 8 after administration of different doses of wild type strains of Neospora caninum-1 (NC-1) in mice.

[0151] FIG. 3 is a dot plot showing the production of specific IgG against Neospora caninum after administration of different doses of wild type strains of Neospora caninum-1 (NC-1) in mice.

[0152] FIG. 4 is a curve showing the tumor volume of mice inoculated with tumor cells and treated with wild type strains of Neospora caninum-1 (NC-1) compared to untreated mice inoculated with the tumor only (control).

[0153] FIG. 5 is a dot plot showing the tumor volume of mice inoculated with tumor cells and treated with wild type strains of Neospora caninum-1 (NC-1) compared to untreated mice inoculated with the tumor only (control) at day 25. **: p-value<0.01 (Kruskall Wallis test).

[0154] FIG. 6 is a dot plot showing the serum level of IFNgamma before or 4 days post-infection (4 DPI) with wild type strains of Neospora caninum-1 in mice implanted with the tumor.

[0155] FIG. 7 is a dot plot showing the in vitro IFNgamma production by spleen lymphocytes recovered from mice treated with wild type strains of Neospora caninum-1 (NC-1) or untreated (Control) and stimulated with concanavalin A (ConA).

[0156] FIG. 8 is an immunoblot showing GRA15II protein expression (visualized by anti-Tag HA). The black arrow indicates the GRA15II protein expected band at 60 kDa molecular weight. Lane 1: Neospora caninum parasites electroporated with a plasmid encoding GRA15II. Lane 2: untransduced Neospora caninum parasites. Lane 3: molecular weights (MW) standards: 17, 26, 42, 55, 72, 95 and 140 kDa.

EXAMPLES

Example 1: In Vivo Effect of Neospora caninum

[0157] Materials and Methods

[0158] Mice

[0159] Eight week-old female inbred C57BL/6 mice are maintained under pathogen-free conditions.

[0160] Neospora caninum-1 Strain

[0161] Tachyzoites of the NC-1 strain of Neospora caninum are harvested from infected human foreskin fibroblasts Hs 27 (ATCC CRL-1634) cultured in monolayers in DMEM, supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin/50 g/ml streptomycin, and 1% HEPES. Neospora caninum tachyzoites may be harvested when monolayers of human foreskin fibroblasts are completely lysed.

[0162] Tumor Cells

[0163] EG7 cells (EL4-OVA thymoma cells transfected with chicken albumin cDNA) are cultured, for example, in RPMI medium, with 510.sup.5 M of 2-mercaptoethanol, 50 UI/mL of penicillin and 50 mg/mL of streptomycin.

[0164] Tumor Cell Inoculations

[0165] 110.sup.5 live EG7 cells are inoculated intradermally in the right flank of the mice. Tumor diameters are measured 3 times weekly, and mice are euthanized when tumor diameters reached 25.000 mm.sup.3.

[0166] Neospora caninum-1 (NC-1) Administration

[0167] Mice are injected subcutaneously in the right flank at day 4 and again at day 7 with 510.sup.6 freshly isolated tachyzoites of NC-1 strain of Neospora caninum.

[0168] Cell Culture Conditions and Cytokine Quantification

[0169] Spleens are harvested and pressed through a stainless steel mesh. Single cell suspensions are obtained by filtration through a nylon mesh to remove tissue debris. Spleen erythrocytes are lysed by hypotonic shock, single cells of spleen are resuspended in RPMI 1640 supplemented with 5% FCS, HEPES (25 mM), L-glutamine (2 mM), sodium pyruvate (1 mM), -mercaptoethanol (510.sup.5 M) and penicillin (50 U/ml)/streptomycin (50 g/ml). Spleen cells are cultured in 96-well plates at 510.sup.5 cells per well, in 200 l of culture medium, alone or containing concanavalin A (10 g/ml). The plates are incubated for 72 hours in 5% CO2 at 37 C. Cell culture supernatants are harvested and kept at 20 C. IFN-gamma quantification is determined by an ELISA assay on the supernatant of the spleen lymphocytes.

[0170] Results

[0171] Neospora caninum Infection is Lethal at Very High Inoculum Doses

[0172] Strains of Neospora caninum challenging study reveals that all mice immunized with 510.sup.6, 510.sup.5, 510.sup.4, and 510.sup.3 of NC-1 tachyzoites survive up to 25 days. One on three mice infected with 510.sup.7 NC-1 tachyzoites succumbs on day 5 (FIG. 1).

[0173] Neospora caninum Induces Humoral and Cellular Immune Responses

[0174] High levels of seric IFN-gamma (approximately 2000 pg/mL) are obtained at day 8 post-infection whatever the doses of injected NC-1 tachyzoites. IFN-gamma secretion at day 2 post-infection is only observed in serum of mice infected with 510.sup.7 and 510.sup.6 NC-1 tachyzoites (FIG. 2).

[0175] All infected mice develop Neospora-specific humoral immune responses at day 21 post-infection. As shown in FIG. 3, highest levels of humoral antibodies are achieved in mice infected with 510.sup.7 and 510.sup.6 NC-1 tachyzoites (approximately 0.8 for a dilution 1/50) (FIG. 3).

[0176] According to these results showing a correlation between the dose and the anti-Neospora immune response, it was decided to focus on the concentration of 510.sup.6 NC-1 tachyzoites for the next experiments.

[0177] Neospora caninum Treatment Suppresses and/or Regresses an Established Solid Tumor Development

[0178] Mice that received EG7 cells develop large tumors (8090507 mm.sup.3) at day 25 post transfer. In contrast, pretreatment with NC-1 tachyzoites significantly reduces the tumor volume (1816465 mm.sup.3), demonstrating that NC-1 tachyzoites suppress the thymome tumor development (FIGS. 4 and 5).

[0179] Neospora caninum Induces a Protective Immune Response Against Tumor Development

[0180] An increase of serum level of IFN-gamma 8 days post tumor implantation and 4 days after the first Neospora dose inoculation (4 DPI) is observed in Neospora-treated mice compared to serum level of IFN-gamma in untreated mice (FIG. 6).

[0181] An increase of IFN-gamma production is observed in vitro by spleen lymphocytes recovered from Neospora-treated mice (NC-1) and stimulated with concanavalin A compared to stimulated spleen lymphocytes recovered from untreated mice (control). This result suggests that Neospora caninum generates a therapeutic antitumor immune response by reversing spleen immunosuppression during tumor development (FIG. 7).

Example 2: Neospora caninum Over-Expressing GRA15II

[0182] In order to obtain a Neospora caninum strain overexpressing GRA15, the sequence encoding GRA15II (strain Me49, sequence ToxoDB, Gene ID: 7895856, TGME49_275470) including a HA-Tag at the C-terminal end was cloned in a vector pUC8 at the PmeI site. The vector pUC8 contains 2 expression cassettes. One expression cassette encodes the chloramphenicol resistance gene in fusion with the GFP protein under the control of the pTUB5 promoter. The second expression cassette encodes the protein of interest, GRA15II, under the control of the pTUB8 promoter whose sequence includes the Kozak sequence, the translation initiation codon and the stop codon.

[0183] Neospora caninum NC-1 parasites (10.sup.7 parasites), taken up in Cytomix medium (120 mM KCl; 5 mM MgCl.sub.2; Hepes 25 mM; 10 mM K.sub.2HPO4/KH.sub.2PO4, pH 7.6; 2 mM EDTA; 0.15 mM CaCl.sub.2, pH 7.6 adjusted with KOH), containing 3 mM ATP and 3 mM Glutathione were then electroporated with 50 g of recombinant plasmid pUC8 GRA15II previously linearized with the PciI restriction enzyme.

[0184] Then, parasites cultured on Human Foreskin Fibroblasts (HFF cells) were selected with a medium containing chloramphenicol. After eight weeks of selection, the chloramphenicol-resistant parasites were analyzed by immunoblotting to visualize the expression of the GRA15II protein.

[0185] The expression of GRA15II protein was analyzed by electrophoresis (5.10.sup.6 parasites per well) followed by a transfer on nitrocellulose membrane. Briefly, parasites were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, sonicated, heated at 100 C. for 3 min, and separated on a polyacrylamide gel. After electrophoresis, proteins were transferred onto a nitrocellulose membrane, which was probed with anti-HA Tag Polyclonal Antibody (71-5500-Invitrogen). Bound antibodies were detected using anti-rabbit immunoglobulin G (IgG, whole molecule)-alkaline phosphatase conjugate (A3687-Sigma-Aldrich). Alkaline phosphatase activity was detected using the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) liquid substrate system (S3771-Promega). Molecular masses standards (prestained SDS-PAGE standards, (ProSieve QuadColor Protein Markers, Lon00193837-Ozyme)) were used.

[0186] The protein is expected at 60 Kda molecular weight. A band indicated by the black arrow is visualized in Lane 1 at approximately 60 Kda (FIG. 8) showing the GRA15II protein expression in Neospora caninum electroporated with the PUC8 GRA15II plasmid in comparison with non-electroporated Neospora caninum in Lane 2 (FIG. 8) showing no band at 60 kda. Bands visualized above the indicated MW may correspond to a dimerized form of the GRA15II protein.