USE OF LIGANDS DERIVED FROM RECEPTOR-BINDING DOMAIN OF PORCINE ENDOGENOUS RETROVIRUS TYPE B FOR DIAGNOSING SMVT-RELATED DISEASES

Abstract

Methods for detecting and/or measuring the level of sodium-dependent multivitamin transporter (SMVT) in a biological sample. The methods include the steps of: (a) contacting the biological sample with a PERV-B.RBD ligand, a variant and/or a fragment thereof; and, detecting and/or measuring the binding of the PERV-B.RBD ligand, variant and/or fragment thereof to SMVT.

Claims

1-14. (canceled)

15. An in vitro method for detecting and/or measuring the level of sodium-dependent multivitamin transporter (SMVT) in a biological sample, wherein said method comprises the steps of: a. contacting said biological sample with a PERV-B.RBD ligand, a variant and/or a fragment thereof; and, b. detecting and/or measuring the binding of said PERV-B.RBD ligand, variant and/or fragment thereof to SMVT.

16. The in vitro method according to claim 15, wherein said method further comprises comparing the binding level measured at step b. with a reference value.

17. The in vitro method according to claim 15, wherein the amino acid sequence of said PERV-B.RBD ligand, variant and/or fragment thereof comprises or consists of an amino acid sequence selected from the group comprising or consisting of the amino acid sequences SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26 and, variants and/or fragments thereof.

18. The in vitro method according to claim 15, wherein said PERV-B.RBD ligand, variant and/or fragment thereof is labeled with a detectable label.

19. The in vitro method according to claim 15, wherein said method is for diagnosing a SMVT-related disease in a subject or for identifying a subject as being at risk of developing a SMVT-related disease.

20. The in vitro method according to claim 19, wherein said SMVT-related disease is cancer.

21. The in vitro method according to claim 19, wherein said SMVT-related disease is a neuroinflammatory disease.

22. The in vitro method according to claim 19, wherein said SMVT-related disease is a disorder of pregnancy.

23. The in vitro method according to claim 19, wherein said SMVT-related disease is an infectious disease.

24. The in vivo method according to claim 15, for use in an in vivo diagnosis method of a SMVT-related disease in a subject.

25. The in vivo method according to claim 24, wherein said SMVT-related disease is a cancer, a neuroinflammatory or neurodegenerative disease, an infectious disease, a disorder of pregnancy or a disorder related to a mutation in the SLC5A6 gene.

26. The in vivo method according to claim 24, wherein said in vivo diagnosis method is based on medical imaging.

27. A PERV-B.RBD ligand, a variant and/or a fragment thereof, coupled to a detectable label.

28. A probe for medical imagery comprising the labeled PERV-B.RBD of claim 27.

29. An in vivo method for detecting and/or measuring the level of sodium-dependent multivitamin transporter (SMVT) in a cell, sample, tissue or organ within a subject, wherein said method comprises the steps of: a. contacting at least one PERV-B.RBD ligand, variant and/or fragment thereof with said cell, sample, tissue or organ within said subject, and b. detecting and/or quantifying the at least one PERV-B.RBD ligand, variant and/or fragment thereof bound to SMVT present in the cell, sample, tissue or organ within said subject.

30. The in vitro method according to claim 20, wherein said cancer is selected from the group consisting of liver cancer, prostate cancer, lung, cancer, gallbladder cancer, pancreas cancer, colon cancer, ovarian cancer, brain cancer, kidney cancer, thyroid cancer and lymphomas.

31. The in vitro method according to claim 21, wherein said neuroinflammatory disease is multiple sclerosis.

32. The in vitro method according to claim 22, wherein said disorder of pregnancy is pre-eclampsia or intrauterine growth retardation.

33. The in vitro method according to claim 23, wherein said infectious disease is caused by a bacterium.

34. The in vitro method according to claim 33, wherein said infectious disease is caused by a bacterium belonging to the order Enterobacteriales.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0298] FIG. 1 is a plot showing a representative example of the result of three independent automated screenings identifying SLC5A6/SMVT as the PERV-B.RBD cognate receptor among 172 soluble carrier family member. The fluorescence intensity (y-axis—positively correlating with the binding of PERV-B.RBD) is plotted for each of the SLC receptor tested (x-axis).

[0299] FIG. 2 is a set of graphs showing the specific PERV-B.RBD binding to SLC5A6/SMVT. A) HEK 293T cells were transfected with empty vector (pCHIX), a SMVT expression or a glucose transporter type 1 (GLUT-1) expression vector. B) HEK 293T cells were transfected with siRNAs directed against luciferase (siLUC, upper panels), SMVT (siSMVT) alone (central panels) or in combination with SMVT expression vector (siSMVT+SMVT, lower panels). SMVT expression level was monitored using PERV-B.RBD ligand (and GLUT-1 expression using human T-cell leukemia virus type 2 (HTLV2)-RBD (H2-RBD) by flow cytometry. Nonspecific staining (filled histograms) and specific binding (solid line histograms) are represented. The number of cells (count—y-axis) is plotted as a function of the fluorescence intensity (AU—x-axis). Relative specific binding of each RDB in the different conditions, expressed as the delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining, is indicated in the upper right of each panel.

[0300] FIG. 3 is a set of graphs confirming the specific PERV-B.RBD binding to SLC5A6/SMVT using a second siSMVT siRNA. HEK 293T cells were transfected with either a siLUC control siRNA (siLUC) (upper panel), or an anti-SMVT siRNA (siSMVT) (lower panel). Variation of relative binding of different RBD ligands was compared by flow cytometry in these two conditions. RBD ligands included a control supernatant preparation with no RBD (MOCK), or a preparation of either PERV-B.RBD (PERV-B), or a RBD from a xenotropic murine retrovirus (XRBD), whose receptor is known to be XPR1/SLC53A1, or the HTLV2-RBD (H2), whose receptor is known to be GLUT1/SLC2A1, or a RBD derived from the bovine leukemia virus (BLV), whose receptor is known to be CAT1/SLC7A1. The number of cells (count—y-axis) is plotted as a function of the fluorescence intensity (AU—x-axis).

EXAMPLES

[0301] The present invention is further illustrated by the following examples.

Example 1: PERVB-RBD Automated Screen Identifies SLC5A6 as the PERVB-RBD Cognate Receptor

[0302] Materials and Methods

[0303] Three independent screenings for PERV-B-RBD (SEQ ID NO: 13) binding on a collection of 172 member of the Solute Carrier protein (SLC) family were performed with a Freedom-EVO® robot from Tecan with acquisition of images and fluorescence intensity parameters by Cellomics (Array Scan XTI HCS Thermo Scientific).

[0304] 20 000 quail QT6 cells per well were seeded in a 96 well plate coated with poly-D-lysine. 24 hours later, cells were transfected with 100 ng of SLC expression vectors using JetPrime transfection reagent (Polypus Transfection 114-15) according to the manufacturer's instructions. QT6 cell were chosen as the spontaneous binding of the PERV-B.RBD ligand was the lowest among the cell lines considered and tested. Cells were washed the next day with PBS and incubated in fresh DMEM/FBS for 48 hours before binding assays. Binding assays were performed on transfected adherent cells that were washed with PBA (PBS with 2% FBS), incubated in 40 μl with either a saturating concentration of the PERV-B RBD ligand fused to a mouse Fc fragment (SEQ ID NO: 13), or a control supernatant, or a control RBD known to interact with a cognate SLC protein. Incubation was carried on for 30 min at 37° C., before cells were washed twice with PBA, incubated with Alexa488-conjugated (Invitrogen) anti-mouse IgG1 antibody (1/500 dilution) at room temperature, washed twice with PBA and fixed with 1% paraformaldehyde (PFA). Transfection and binding assays were performed on the Freedom EVO robot (Tecan). Images and fluorescence intensity parameters were acquired by Cellomics (Array Scan XTI HCS, Thermo Scientific). Fluorescent signal was analyzed using GraphPad Prism 5.

[0305] Results and Conclusions

[0306] Among the collection of members of the Solute carrier (SLC) protein family, PERV-B.RBD ligand binds to SMVT/SLC5A6 (FIG. 1).

Example 2: Specificity of the Binding of PERV-B.RBD Ligand to SMVT/SLC5A6

[0307] Material and Methods

[0308] 5×10.sup.5 HEK293T cells were seeded on a 6-well plate and transfected with a siRNA directed either against the firefly luciferase gene (siLUC, 5′-CUUACGCUGAGUACUUCGA-3′—SEQ ID NO: 15), or a siRNA directed against the SLC5A6 gene (siSMVT, 5′-GGAUGAGUCUUGGUGUGUUTT-3′—SEQ ID NO: 16). Forty-eight hours post-transfection, cells were detached with 1 mM EDTA in PBS. For the binding assay, 1×10.sup.5 cells were resuspended in 100 μl of PBA (PBS with 2%1-BS) containing a saturating concentration of either the PERV-B.RBD (SEQ ID NO: 13), or the human T-cell leukemia virus type 2 (HTLV2)-RBD known to bind SLC2A1/GLUT1, and incubated at 37° C. for 30 min Cells were then washed twice with 100 μl of PBA, incubated with Alexa488-congugated anti-mouse IgG1 at 4° C. for 20 min, washed twice and resuspended in 200 μl of PBA. The results were acquired by flow cytometry on a FACSCalibur (Becton Dickinson) and analyzed by FlowJo. Nonspecific staining with no RBD (filled histograms) and specific binding of the RBD (solid line histograms) are represented as delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining. HEK 293T cells were transfected as above with either the empty vector (pCHIX, “vecteur vide”), the SMVT expression vector (SMVT), or the GLUT-1 expression vector. All were tested with PERV-B.RBD (SEQ ID NO: 13) or HTLV2-RBD, the latter known to bind SLC2A1/GLUTE

[0309] Results and Conclusion

[0310] The binding of the PERV-B.RBD ligand increased specifically when cells overexpressed SMVT (FIG. 2A) and decreased specifically in cell underexpressing SMVT (FIG. 2B). The PERV-B.RBD thus binds specifically to SMVT/SLC5A6, allowing it use in the diagnosis of disease characterized by a variation of SMVT expression.

Example 3: Specificity of the Binding of PERV-B.RBD Ligand to SMVT/SLC5A6, as Assessed with a Second Anti-SMVT siRNA

[0311] Material and Methods

[0312] 5×10.sup.5 HEK293T cells were seeded on a 6-well plate and transfected with a siRNA directed either against the firefly luciferase gene (siLUC, 5′-CUUACGCUGAGUACUUCGA-3′—SEQ ID NO: 15), or a siRNA directed against the SLC5A6 gene and different from the siRNA used in Example 2 (siSMVT A, 5′GCAGGAUCAUGCCAGAAAUTT-3′—SEQ ID NO: 23). Forty-eight hours post-transfection, cells were detached with 1 mM EDTA in PBS. For the binding assay, 1×10.sup.5 cells were resuspended in 100 μl of PBA (PBS with 2% FBS) containing a predetermined saturating concentration of either the PERV-B.RBD (SEQ ID NO: 13), or a RBD from a xenotropic murine retrovirus (XRBD), whose receptor is known to be XPR1/SLC53A1, or the HTLV2-RBD (H2), whose receptor is known to be GLUT1/SLC2A1, or a RBD derived from the bovine leukemia virus (BLV), whose receptor is known to be CAT1/SLC7A1, or a control supernatant preparation with no RBD (MOCK), followed by an incubation at 37° C. for 30 min. All RBD were produced as fusion proteins with mFC (PERV-B.RBD, XRBD, BLV-RBD and HTLV2-RBD). Cells were then washed twice with 100 μl of PBA, incubated with Alexa488-congugated anti-mouse IgG1 at 4° C. for 20 min, and washed twice and resuspended in 200 μl of PBA. The results were acquired by flow cytometry on a FACSCalibur (Becton Dickinson) and analyzed by FlowJo. Relative specific binding of each RDB in the different conditions, expressed as the delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining.

[0313] Results and Conclusion

[0314] Specific binding of the PERV-B.RBD ligand decreased significantly when cells where treated with anti-SMVT siRNA, with a drop of over 2-fold (77.5 to 34, FIG. 3 and table 1). Specific drop of PERV-B.RBD binding observed upon introduction of siSMVT was assessed as binding of XRBD, H2-RBD or BLV-RBD did not vary significantly between siLUC (FIG. 3 left panel) and siSMVT treatment (FIG. 3 right panel). This confirmed that PERV-B.RBD binds specifically to SMVT/SLC5A6, allowing it use in the diagnosis of disease characterized by a variation of SMVT expression.

TABLE-US-00021 TABLE 1 Relative binding of each RBD preparation and the control (MOCK) is expressed Relative specific binding of each RDB in the different conditions, expressed as the delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining. Ligand siLUC siSMVT A MOCK 2.6 2.2 PERV-B.RBD 77.5 34.0 XRBD 12.6 14.6 HTLV2-RBD 18.3 21.3 BLV-RBD 110.0 110.0

Example 4: PERV-B.RBD Ligand Allows the Detection of Variations of the Expression of SMVT at the Surface of Cancer Cells

[0315] Materials and Methods

[0316] For the binding assay, 1×10.sup.5 cells of each cell type were resuspended in 100 μl of PBA (cell density of 1×10.sup.6 per mL) containing a saturating concentration of either the PERV-B-RBD (SEQ ID NO: 13), and incubated at 37° C. for 30 min. Cells were washed twice with 100 μl of PBA, incubated with PE-conjugated anti-mouse IgG1 at 4° C. for 20 min, washed twice and resuspended in 200 μl of PBA. The results were acquired by flow cytometry on a FACS verse (Becton Dickinson) and analyzed with the FlowJo software (FLOWJO, LLC). Relative specific binding of PERV-B.RBD to the different cells is expressed as the delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining.

[0317] Results and Conclusions

[0318] The binding of PERV-B.RBD to the surface of the cells was expressed in terms of the mean signal to noise ratio, for each cell type. Most cancer cell lines displayed a signal to noise ratio over 100 (table 2).

[0319] The results indicate that variations of expression level of SMVT, are indicative of cancer.

TABLE-US-00022 TABLE 2 Expression of SMVT in different human cancer cell lines. Bold font distinguishes cells of cancerous origin. Relative specific binding of PERV-B.RBD to the different cells is expressed as the delta of the geometric mean of the fluorescence intensity between a specific binding and a nonspecific staining. Specific PERV- Cell type B.RBD binding RS4; 11 (lymphoma cell line, B-type) 6 Cardiomyocytes (ES-derived) 12 Primary hepatocytes 14 hMSC (hES-derived) 16 IC8LC10 (Lung, NSCLC cell line) 16 Circulating granulocytes 20 HID28-1 (Prostate, adenocarcinoma cell line) 30 RCC-49 (Kidney, carcinoma cell line) 37 SU-DHL6 (lymphoma cell line) 47 GBM14-CHA (Brain, gliobastoma cell line) 55 HEK-293T 64 Red blood cells 70 Circulating lymphocytes 81 Circulating monocytes 106 OVA2-BUR (Ovarian - Adenocarcinoma cell line) 111 KP4 (Pancreas, ductal cell carcinoma cell line) 118 T84 (colorectal carcinoma cell line) 120 MIA PaCa-2 (Pancreas, carcinoma cell line) 173 TC122a (Colon - carcinoma cell line) 175 AsPC1 (Pancreas, adenocarcinoma cell line) 202 Mz-ChA-1 (Gallbladder, carcinoma cell line) 242 Mz-ChA-2 (Gallbladder, carcinoma cell line) 248 IC20-DAN (Lung, NSCLC cell line) 250 HID28-2 (Prostate, adenocarcinoma cell line) 266 HepG2 (Liver, hepatocellular carcinoma cell line) 343