Dendritic-marker expressing macrophage cultures and methods...

Details Dendritic-marker expressing macrophage cultures and methods... Dendritic-marker expressing macrophage cultures and methods...

1998-09-30

2001-05-01

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S091500, C435S325000, C435S373000

Reexamination Certificate

active

06225048

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides methods of culturing virus in monocyte-derived macrophages (“MDM”) through allogeneic stimulation of peripheral blood mononuclear cells (“PBMC”) or through culture of PBMC with media comprising IFN&ggr;, stable cultures of such virally permissive monocyte-derived macrophages, and methods of screening for viral inhibitors using such cultures.
BACKGROUND OF THE INVENTION
Human cytomegalovirus (“HCMV”) is a ubiquitous pathogen that is the major cause of morbidity and mortality in immunocompromised individuals, such as transplant and AIDS patients, as well as a leading cause of congenital birth defects (Britt et al., in
Fields Virology
, pp. 2493-2523 (Fields et al., eds. 1996)). HCMV is also associated with the development of atherosclerosis, restenosis after coronary angioplasty, chronic rejection in organ transplant patients (Grattan et al.,
Jama
261:3561-3566 (1989); Melnick et al.,
Bioessays
17:899-903 (1995); Zhou et al.,
NEJM
335:624-630 (1996)) and chronic graft-versus-host disease in bone marrow transplant patients (Lönnqvist et al.,
Transplantation
38:465-468 (1984); Söderberg et al.,
Transplantation
61:600-609 (1996)). Most individuals become infected with HCMV early in life, and depending on the geographic location, between 60-100% of adults are carriers of the virus (Britt et al., in
Fields Virology
pp. 2493-2523 (Fields et al., eds. 1996)).
Similar to other herpesviruses, HCMV establishes life-long latency in the host after a primary infection, which is characterized by persistence of the viral genome without the production of infectious virus. The respective sites of latency for other herpesviruses such as Epstein Barr and herpes simplex viruses are B cells and neurons (Kieff, in
Fields Virology
pp. 2343-2396 (Fields et al., eds. 1996); Roizman et al., in
Fields Virology
pp. 2231-2296 (Fields et al., eds. 1996)). However, although transmission of latent HCMV has been shown to occur through transfusion of blood products, bone marrow grafts, and solid organs (Britt et al., in
Fields Virology
pp. 2493-2523 (Fields et al., eds. 1996); Chou,
NEJM
314:1418-1423 (1986); Meyers,
Am. J. Med.
81:27-38 (1986); Tegtmeier,
Arch. Pathol. Lab. Med.
113:236-245 (1989)), the identity of cells harboring latent or persistent virus is unknown. In addition to HCMV, the identity of cells harboring other viruses is unclear, and methods of culturing such viruses are unknown.
Several animal models have been established to understand mechanisms involved in latency and reactivation of CMV (Bruning et al.,
Transplantation
41:695-698 (1986); Hamilton et al.,
Transplantation
39:290-296 (1985); Reddehase et al.,
J. Exp. Med.
179:185-193 (1994); Yagyu et al.,
Transpl. Proc.
25:1152-1154 (1993)). In murine organ transplant models, reactivation of murine cytomegalovirus (“MCMV”) was shown to be influenced by the state of imrnmunosuppression and histoincompatibility between the donor and the recipient (Bruning et al.,
Transplantation
41:695-698 (1986); Hamilton et al.,
Transplantation
39:290-296 (1985); Reddehase et al.,
J. Exp. Med.
179:185-93 (1994); Yagyu et al.,
Transpl. Proc.
25:1152-1154 (1993)). In MCMV latently-infected mice, the spleen, kidneys, and bone marrow were shown to be important sources of virus (Jordan et al.,
J. Clin. Invest.
70:762-768 (1982); Mercer et al.,
J. Virol.
62:987-997 (1988); Olding et al.,
J. Exp. Med.
141:561-572 (1975)). Activation of virus in latently infected animals has been shown to occur through either intraperitoneal injection of thioglycollate (Pollock et al.,
Virology
227:168-179 (1997)) or allogeneic stimulation (Schmader et al.,
J. Inf. Dis.
166:1403-1407 (1992)). The peripheral blood of latently infected animals was also demonstrated to be a reservoir of virus since allogeneic stimulation resulted in the activation of MCMV replication (Schmader et al.,
J. Inf. Dis.
166:1403-1407 (1992); Olding et al.,
J. Exp. Med.
141:561-572 (1975); Jordan et al.,
J. Clin. Invest.
70:762-876 (1982); Mercer, et al.,
J. Virol.
62:987-997 (1988); Koffron et al.,
Scand. J. Inf. Dis.
-
Suppl.
99:612 (1995); Stoddart et al.,
J. Virol.
68:6243-6253 (1994); Pollock et al.,
Virology
227
:
168
-
179
(1997)).
In humans, examination of organ tissues and peripheral blood obtained from patients with HCMV disease has suggested that PBMC are a viral reservoir of HCMV (Chou,
NEJM
314:1418-1423 (1986); Meyers,
Am. J. Med.
81:27-38 (1986); Taylor-Wiedeman, et al.,
J. Gen. Virol.
72:2059-2064 (1991); Tegtmeier,
Arch. Pathol. Lab. Med.
113:236-245 (1989); Gnann et al.,
Am. J. Pathol.
132:239-248 (1988)). Further analyses of separated PBMC populations obtained from HCMV-seropositive donors have identified monocytes as the predominant infected cell type (Taylor-Wiedeman et al.,
J. Gen. Virol.
72:2059-2064 (1991)). While viral replication in monocytes is restricted to early events of gene expression (Ibanez et al.,
J. Virol.
65:6581-6588 (1991)), examination of organ tissues early in HCMV disease has demonstrated extensive viral gene expression in tissue macrophages (Gnann et al.,
Am. J. Pathol.
132:239-248 (1988); Sinzger et al.,
J. lnf. Dis.
173:240-245 (1996)).
CD14
+
monocytes in the peripheral blood are terminally differentiated cells derived from myeloid/granulocyte precursors. In vivo, stimulation of monocytes by contact with T and B cells during antigen processing events induces differentiation of monocytes into macrophages for function as immune effector cells. A variety of tissue culture protocols have been established to mimic the in vivo development of monocyte-derived macrophages (“MDM”), which includes treatment of monocytes with cytokines, mitogens, corticosteroids, or lipopolysaccharide (“LPS”) (reviewed by Adams et al., in
The Macrophage
pp. 77-115 (Lewis et al., eds. 1992)). Caux et al. report, using FACS sorting, that a transient population of CD14
+
, CD1a
+
cells represent a developmental phase in MDM differentiation (Caux et al.,
J. Exp. Med.
184:695-706 (1996)). MDM derived by these methods have been used for the in vitro propagation of certain macrophage-tropic viruses (Gendelman et al.
J. Exp. Med.
167:1428-1441 (1988); Matloubian et al.,
J. Virol.
67:7340-7349 (1993); Schrier et al.,
J. Virol.
64:3280-3288 (1990)). Rettig et al. identify endogenous CD68
+
, CD83
+
bone marrow stromal cells that are infected with HHV8 (Rettig et al.,
Science
276:1851-1854 (1997)). However, culture of HCMV in MDM has proven to be difficult, often resulting in abortive infection (Rice et al.,
Proc. Natl. Acad. Sci
. (
USA
) 81:6134-6138 (1984); Taylor-Wiedeman et al.,
J. Virol.
68:1597-1604 (1994)).
MDM differentiation systems have also been developed that rely on the mitogenic stimulation of PBMC to generate HCMV permissive macrophages (Ibanez et al.,
J. Virol.
65:6581-6588 (1991)). Although macrophages differentiated by this method are susceptible to in vitro, exogenous HCMV infection, attempts to reactivate HCMV from PBMC obtained from latently infected individuals have been unsuccessful using this method or others (Taylor-Wiedeman et al.,
J. Virol.
68:1597-1604 (1994)). Thus, there is a need to identify the specific cellular reservoir of latent HCMV infection, to isolate cultures of such cells, and to establish methods of culturing cells in which HCMV replicates, where the cells are latently infected with HCMV and/or additional viruses, or can be infected in vitro.
SUMMARY OF THE INVENTION
The present invention identifies for the first time the specific cellular reservoir of latent viral, e.g., HCMV infection. The invention also provides methods of reactivating HCMV and other viruses in long-term cultures of allogeneically-stimulated monocyte-derived macrophages (“MDM”). Infectious virus was recovered from a myeloid lineage cell phenotype of MDMs that expressed both macrophage (CD14) and dendritic cell markers (CD83). In addition, these cells are permissive for exogenous viral infection. These observati

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