Dendritic-marker expressing macrophage cultures and methods of reactivating latent virus
AssigneeOregon Health Sciences University
InventorsCecilia E. Soderberg-Naucler, Kenneth N. Fish, Ashlee Moses, Daniel Streblow, Jay Nelson
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Abstract
The present invention provides methods of latent virus reactivation in monocyte-derived macrophages through allogeneic stimulation of peripheral blood mononuclear cells (“PBMC”), methods of culturing virus, and cultures of virally permissive monocyte-derived macrophages.
Claims80
A method of replicating viruses in virally permissive monocyte-derived macrophages, the method comprising the steps of:
- (a) culturing monocytes under conditions where the monocytes are in fluid communication with viable, allogeneically stimulated peripheral blood mononuclear cells (PBMC) to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- and
- (b) permitting viral replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture, and wherein the monocyte derived-macrophages have a majority population of cells bearing CD83 and CD14.
A method for screening for inhibitors of virus production using virally permissive monocyte-derived macrophages, the method comprising the steps of:
- (a) culturing monocytes under conditions where the monocytes are in fluid communication with viable, allogeneically stimulated peripheral blood mononuclear cells (PBMC) to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- (b) permitting viral replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture;
- (c) contacting the monocyte-derived macrophages of step (b) with substances suspected of having the ability to inhibit viral production;
- and
- (d) detecting the level of virus production in the monocytes-derived macrophages.
A stable culture of virally permissive monocyte-derived macrophages, wherein the monocyte-derived macrophages are derived from monocytes exposed to allogeneically stimulated peripheral blood mononuclear cells (PBMC) for a time sufficient to and a concentration sufficient to:
- (i) stimulate active differentiation of the monocytes into monocyte-derived macrophages, and (ii) stimulate viral production in the monocyte-derived macrophages;
- and, wherein the virally permissive monocyte-derived macrophages produce at least 10,000 fold greater virus than non-allogeneically stimulated monocytes, and wherein the monoczte derived-macrophages have a majority population of cells bearing CD83 and CD14.
A stable culture of virally permissive monocyte-derived macrophages having a population defined as at least 85% bearing CD83 and CD14.
A method of culturing virally permissive monocyte-derived macrophages, the method comprising the step of culturing monocytes under conditions where the monocytes are in fluid communication with viable, allogeneically stimulated peripheral blood mononuclear cells (PBMC) to activate the monocytes to differentiate into monocyte-derived macrophages, wherein the monocyte derived-macrophages have a majority population of cells bearing CD83 and CD14.
A stable culture of virally permissive monocyte-derived macrophages having the following characteristics:
- (i) comprising dendritic cell markers CD68, CD83, and CD1a;
- (ii) comprising macrophage cell markers CD64 and CD14;
- and
- (iii) derived from CD14+ monocytes.
A method of replicating viruses in virally permissive monocyte-derived macrophages, the method comprising the steps of:
- (a) culturing CD14+ monocytes under conditions where the monocytes are exposed to conditioned media comprising IFNγ in an amount sufficient to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- and
- (b) permitting viral replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture, and wherein the monocyte derived-macrophages have a majority population of cells bearing CD83 and CD14.
A method for screening for inhibitors of virus production using virally permissive monocyte-derived macrophages, the method comprising the steps of:
- (a) culturing CD 14+ monocytes under conditions where the monocytes exposed to conditioned media comprising IFNγ in an amount sufficient to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- (b) permitting viral replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture;
- (c) contacting the monocyte-derived macrophages of step (b) with substances suspected of having the ability to inhibit viral production;
- and
- (d) detecting the level of virus production in the monocytes-derived macrophages.
A stable culture of virally permissive monocyte-derived macrophages, wherein the monocyte-derived macrophages are derived from CD14+ monocytes exposed to conditioned media comprising IFNγ in an amount sufficient to and for a time sufficient to:
- (i) stimulate active differentiation of the CD14+ monocytes into monocyte-derived macrophages, and (ii) stimulate viral production in the monocyte-derived macrophages;
- and, wherein the virally permissive monocyte-derived macrophages produce at least 10,000 fold greater virus than non-allogeneically stimulated monocytes.
A stable culture of virally permissive monocyte-derived macrophages having a population defined as at least 85% bearing CD83 and CD14.
A method of culturing virally permissive monocyte-derived macrophages, the method comprising the step of culturing CD14+ monocytes under conditions where the monocytes are exposed to conditioned media comprising IFNγ in an amount sufficient to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages, and wherein the monocyte derived-macrophages have a majority population of cells bearing CD83 and CD14.
A method of infecting cells with virus, the method comprising the steps of:
- (a) culturing monocytes under conditions where the monocytes are in fluid communication with viable, allogeneically stimulated peripheral blood mononuclear cells (PBMC) to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- (b) permitting virus replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture;
- (c) collecting virus from the culture;
- and
- (d) contacting a second culture of cells with the virus, thereby infecting the cells with the virus.
A method of infecting cells with virus, the method comprising the steps of:
- (a) culturing CD14+ monocytes under conditions where the monocytes are exposed to conditioned media comprising IFNγ in an amount sufficient to activate the monocytes to differentiate into virally permissive monocyte-derived macrophages;
- (b) permitting viral replication in the monocyte-derived macrophages, where the virus is either latent in the monocytes or exogenously added to the culture.
- (c) collecting virus from the culture;
- and
- (d) contacting a second culture of cells with the virus, thereby infecting the cells with the virus.
Dependencies are inferred from the claim text.