Claims for Patent: 10,435,665

Summary for Patent: 10,435,665

Title:	Method of culturing segmented filamentous bacteria in vitro
Abstract:	The present invention relates to an in vitro method of culturing a segmented filamentous bacterium strain, comprising co-culturing said segmented filamentous bacterium strain with a eukaryotic host cell, wherein the culture is performed at an O.sub.2 level inferior to 5% in a rich tissue culture liquid medium containing bacterial medium components including iron. The present invention also relates to methods for genetically modifying a segmented filamentous bacterium strain comprising a step a culturing the strain in vitro.
Inventor(s):	Gerard; Eberl (Paris, FR), David; Bikard (Paris, FR), Pamela; Schnupf (Paris, FR), Nadine; Cerf Bensussan (Paris, FR), Valerie; Gaboriau-Routhiau (Clamart, FR), Philippe; Sansonetti (Paris, FR)
Assignee:	INSTITUT PASTEUR (Paris, FR) FONDATION IMAGINE (Paris, FR) ASSISTANCE PUBLIQUE--HOPITAUX DE PARIS (Paris, FR) INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) (Paris, FR) UNIVERSITE PARIS DESCARTES (Paris, FR)
Application Number:	15/538,184
Patent Claims:	1. An in vitro method of culturing a segmented filamentous bacterium (SFB) strain, comprising co-culturing said SFB strain with a eukaryotic host cell, wherein the culture is performed at an O.sub.2 level of less than 5% in a rich tissue culture liquid medium comprising bacterial medium components and iron, and wherein the co-culture is performed until the SFB strain releases intracellular offspring or spores. 2. The method according to claim 1, wherein the SFB strain is a wild-type strain or a genetically modified strain. 3. The method according to claim 2, wherein the genetically modified SFB strain expresses an antigen of a diarrheal pathogen selected from the group consisting of Shigella, enterotoxic Escherichia coli, and attaching and effacing lesion (A/E)-inducing enteropathogenic Escherichia coli. 4. The method according to claim 3, wherein the antigen is Shigella IpaB protein or IpaD protein. 5. The method according to claim 3, wherein the antigen is an enterotoxic Escherichia coli antigen selected from colonization factor antigen I, adhesin CfaE, a heat-labile toxin, and nontoxic B subunit of heat-labile toxin. 6. The method according to claim 3, wherein the antigen is an attaching and effacing lesion (A/E)-inducing enteropathogenic Escherichia coli intimin, or a fragment encoding amino adds 363 to 808 of the 94 kDa beta-intimin antigen. 7. The method according to claim 2, wherein the SFB strain is isolated from a mammal. 8. The method according to claim 7, wherein the SFB strain is isolated from a mammal selected from the group consisting of a human and a mouse. 9. An in vitro method of culturing a SFB strain, comprising the steps of: a) growing eukaryotic host cells on a solid culture medium; b) transferring the eukaryotic host cells grown in step a) into a eukaryotic host cell-SFB liquid culture medium to provide a cell culture; c) challenging the cell culture of step b) with a SFB strain; and d) co-culturing the eukaryotic host cells and the SFB strain in the liquid culture medium at an O.sub.2 level of less than 5%, wherein the co-culture is performed until the SFB strain releases intracellular offspring or spores. 10. The method according to claim 9, wherein the eukaryotic host cell is an epithelial cell or a cancer cell. 11. The method according to claim 9, wherein the eukaryotic host cells are gastrointestinal tract cells or carcinoma cells. 12. The method according to claim 9, wherein the eukaryotic host cells are selected from the group consisting of human Caco-2 cell line cells, human TC7 cell line cells, human HeLa cell line cells, mouse mICcl2 cell line cells, and mouse CMT93 cell line cells. 13. The method according to claim 9, wherein in step a) the eukaryotic host cells are grown at an O.sub.2 level of from 0% to 5%. 14. The method according to claim 9, wherein in step a) the eukaryotic host cells are grown at an O.sub.2 level of from 0.5 to 3%. 15. The method according to claim 9, wherein in step a) the eukaryotic host cells are grown at an O.sub.2 level of from 1 to 2.5%. 16. The method according to claim 9, wherein in step b) the eukaryotic host cell-SFB liquid culture medium is a rich tissue culture medium comprising bacterial medium components and iron. 17. The method according to claim 16, wherein the liquid culture medium comprises brain-heart infusion and a yeast/peptone/casein amino-acid mixture. 18. The method according to claim 16, wherein the liquid culture medium is supplemented with from 1% to 5% decomplemented fetal calf serum. 19. The method according to claim 16, wherein the liquid culture medium is supplemented with from 1% to 3% decomplemented fetal calf serum. 20. The method according to claim 16, wherein the liquid culture medium is supplemented with 2% decomplemented fetal calf serum. 21. The method according to claim 16, wherein the liquid culture medium further comprises sugars, retinoic acid and/or nucleotides. 22. The method according to claim 16, wherein the iron is in the form of Fe.sup.2+/Fe.sup.3+, 3.times.Fe.sup.2+, 3.times.Fe.sup.2+ or hemin. 23. The method according to claim 16, wherein the iron concentration is from 0.015 to 0.05 mM. 24. The method according to claim 16, wherein the iron concentration is from 0.02 to 0.04 mM. 25. The method according to claim 9, wherein the liquid culture medium comprises DMEM/F12 advanced medium, supplemented with fetal calf serum, non-essential amino acids, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, brain-heart infusion, a yeast/peptone/casein amino-acid mixture and iron. 26. The method according to claim 9, wherein in step c) the SFB strain is in the form of a filament or an intracellular offspring. 27. The method according to claim 9, wherein in step c), the SFB strain is directly contacted with the eukaryotic host cells. 28. The method according to claim 27, wherein the co-culture of the eukaryotic host cells and the SFB strain is performed at an O.sub.2 level of from 0.5 to 3%. 29. The method according to claim 9, wherein the co-culture of the eukaryotic host cells and the SFB strain is performed at an O.sub.2 level of from 0% to 5%. 30. The method according to claim 9, wherein the co-culture of the eukaryotic host cell and the SFB is performed at an O.sub.2 level of from 1 to 2.5%. 31. The method according to claim 9, wherein iron is at a concentration of from 0.015 to 0.05 mM. 32. The method according to claim 9, further comprising recovering the cultured SFB strain, wherein the cultured SFB strain is recovered in a form of at least one of a filament, an intracellular offspring and a spore. 33. The method according to claim 9, wherein the SFB strain is a wild-type strain or a genetically modified strain. 34. The method of claim 9, wherein the eukaryotic host cells grown in step a) are grown until a cell confluence of at least 20% is obtained. 35. The method of claim 9, wherein the eukaryotic host cells grown in step a) are grown until a cell monolayer is obtained. 36. The method of claim 9, wherein the eukaryotic host cells grown in step a) are plated on a plate. 37. The method of claim 9, wherein the eukaryotic host cells grown in step a) are plated on a tissue culture well or transwell, prior to step b). 38. The method of claim 9, wherein the seeding density of the eukaryotic host cells in a) is between 1.times.10.sup.4 and 6.times.10.sup.4 cells per cm.sup.2. 39. The method according to claim 9, wherein the SFB strain is added in step c) when the culture density of the eukaryotic host cells is from 0.5+10.sup.5 to 3.times.10.sup.5 cells per cm.sup.2. 40. The method according to claim 9, wherein the ratio in step c) between the eukaryotic host cells and the SFB strain is from 0.1 to 100. 41. The method according to claim 40, wherein the ratio between the eukaryotic host cells and the SFB strain is from 0.3 to 60. 42. The method according to claim 9, wherein the co-culture is performed for a duration selected from the group consisting of 1, 2, 3, 4, 5 and 6 days. 43. The method according to claim 9, wherein d) comprises applying conditions to induce spore formation, selected from adding an oxidative stress and short-term culture in the presence of an antibiotic. 44. A food product comprising the recovered SFB strain made by the method of claim 32. 45. The food product according to claim 44, wherein the SFB strain is alive.

Details for Patent 10,435,665

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	PANHEMATIN	hemin for injection	For Injection	101246	07/20/1983		2034-12-23