U.S. patent application number 14/398865 was filed with the patent office on 2015-05-14 for broad antiviral therapy with membrane modifying oxysterols.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Genhong Cheng, Su-Yang Liu.
Application Number | 20150133420 14/398865 |
Document ID | / |
Family ID | 49514946 |
Filed Date | 2015-05-14 |
United States Patent
Application |
20150133420 |
Kind Code |
A1 |
Cheng; Genhong ; et
al. |
May 14, 2015 |
BROAD ANTIVIRAL THERAPY WITH MEMBRANE MODIFYING OXYSTEROLS
Abstract
This invention relates, e.g., to a method for inhibiting the
growth and/or proliferation and/or infectivity of a virus in a
cell, such as a mammalian cell (e.g. for inhibiting entry of the
virus into the cell), comprising administering, or causing to be
administered, to the cell, 25-hydroxycholesterol (25HC) in an
amount sufficient to inhibit the growth and/or proliferation and/or
infectivity of the virus in the cell. The method can be carried out
in vivo or in vitro. Among the viruses that can be inhibited are,
e.g., VSV, HSV, MHV68, HCV, HIV, EBOV, RVFV, RSSEV and Nipah virus.
In one embodiment of the invention, the 25HC is administered
topically, e.g. to a mucosal surface.
Inventors: |
Cheng; Genhong; (Los
Angeles, CA) ; Liu; Su-Yang; (Los Angeles,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
49514946 |
Appl. No.: |
14/398865 |
Filed: |
May 6, 2013 |
PCT Filed: |
May 6, 2013 |
PCT NO: |
PCT/US2013/039748 |
371 Date: |
November 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61643110 |
May 4, 2012 |
|
|
|
Current U.S.
Class: |
514/182 ;
435/375; 435/5 |
Current CPC
Class: |
A61P 31/12 20180101;
A61K 31/575 20130101; A61K 47/40 20130101; A61P 31/00 20180101 |
Class at
Publication: |
514/182 ;
435/375; 435/5 |
International
Class: |
A61K 31/575 20060101
A61K031/575 |
Claims
1. A method for inhibiting the growth and/or proliferation and/or
infectivity of a virus in a cell, comprising administering, or
causing to be administered, to the cell, 25-hydroxycholesterol
(25HC) in an amount sufficient to inhibit the growth and/or
proliferation and/or infectivity of the virus in the cell, wherein,
if the cell is in vitro, the 25HC is administered to the cell, and
the virus is vesicular stomatitis virus (VSV), herpes simplex virus
(HSV), murine gammaherpes virus (MHV68), hepatitis C virus (HCV),
Ebola virus (EBOV), or Nipah virus; and wherein, if the cell is in
a subject, the 25HC is administered or caused to be administered to
the subject, and the virus is vesicular stomatitis virus (VSV),
herpes simplex virus (HSV), murine gammaherpes virus (MHV68),
hepatitis C virus (HCV), human-immunodeficiency virus (HIV), Ebola
virus (EBOV), or Nipah virus.
2. The method of claim 1, wherein the method is a method for
preventing a viral infection of a mammalian cell in vitro, and the
method comprises administering to the mammalian cell 25HC in an
amount sufficient to inhibit infectivity of the virus in the
cell.
3. The method of claim 1, wherein the method is a method for
preventing a viral infection of a mammal, and the method comprises
administering, or causing to be administered, to the mammal, 25HC
in an amount sufficient to inhibit infectivity of the virus in the
mammal.
4. The method of claim 1, wherein the method is a method for
inhibiting entry of the virus into a mammalian cell in vitro, and
the method comprises contacting the mammalian cell with 25HC in an
amount sufficient to inhibit entry of the virus into the cell.
5. The method of claim 1, wherein the method is a method for
inhibiting entry of the virus into a cell in a mammalian subject,
and the method comprises administering, or causing to be
administered, to the mammal, 25HC in an amount sufficient to
inhibit entry of the virus into the cell.
6. The method of claim 3, wherein said 25HC is administered by a
route selected from the group consisting of topical administration,
oral administration, nasal administration, rectal administration,
vaginal administration, intraperitoneal injection, intravascular
injection, subcutaneous injection, transcutaneous administration,
inhalation administration, and intramuscular injection.
7. The method of claim 3, wherein the 25HC is administered
topically, vaginally, rectally, or to the buccal cavity.
8. The method of claim 3, wherein the 25HC is s administered to a
mucosal surface.
9. The method of claim 3, wherein the 25HC is formulated as a
cream, gel, or foam for rectal delivery or vaginal delivery or
topical administration.
10. The method of claim 3, wherein the 25HC is formulated as a
mouthwash for delivery to the buccal cavity.
11. The method of claim 3, wherein the 25HC is formulated for oral
or intravenous delivery.
12. The method of claim 11, wherein the 25HC is solubilized in
(2-hydroxy)-beta-cyclodextrin.
13. The method of claim 2, wherein the mammalian cell is from a
non-human mammal.
14. The method of claim 2, wherein the mammalian cell is human.
15. The method of claim 14, wherein the human is identified as
being at risk for an infection by the virus.
16. The method of claim 14, wherein the human is identified as
having an infection by the virus.
17. A method for identifying putative inhibitors of viral entry
into cells which exhibit lower levels of side effects than does
25HC, comprising testing analogs of 25HC in vitro for their ability
to a) exhibit anti-viral activity, b) exhibit lower levels of cell
cytoxicity than does 25HC, and c) inhibit lipid metabolism to a
lower level than does 25HC.
18. The method of claim 1, which is a method for inhibiting the
growth and/or proliferation and/or infectivity of a virus in a
cell.
Description
[0001] This application claims the benefit of the filing date of
U.S. Provisional application 61/643,110, filed May 4, 2012, which
is incorporated by reference herein in its entirely.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 3, 2013, is named 58086-347965_SL.txt and is 3,313 bytes in
size.
BACKGROUND INFORMATION
[0003] Viruses are obligate intracellular pathogens that--despite
having unique structure and function--undergo lifecycle stages of
entry, replication, protein synthesis, assembly, and egress. Upon
specific binding to cell surface molecules, non-enveloped virus can
enter the cell directly while enveloped viruses undergo fusion
process that requires specific interactions between the viral and
cellular receptors and membranes. After entry, viral components are
released into the cytoplasm and may enter the nucleus. Although
incipient viral proteins may be sufficient to initiate early
lifecycle processes, full viral replication, transcription and
translation require utilization of cellular factors. The newly
synthesized viral proteins and genome are then coordinately
assembled into virions, which then exit the cell by lysis or
budding.
[0004] While viruses exploit host factors to successfully
replicate, the innate immune system produces interferons (IFN),
essential antiviral cytokines that induce wide array of antiviral
effectors. Individually, many of these IFN-stimulated genes (ISGs)
work to inhibit virus at particular stages of its lifecycle. IFITM
proteins block viral entry and ISG20, a 3-5' exonuclease, degrades
single stranded viral RNA; PKR inhibits viral translation through
suppression of eIF2a elongation factors and tetherin prevents
release of virions from the cell (Degols et al., June; Garcia et
al., 2006; Brass et al., 2009; Perez-Caballero et al., 2009). These
ISGs exemplify only a few of the hundreds of confirmed ISGs; most
of them are uncharacterized.
[0005] Cholesterol-25-hydroxylase (Ch25h) is an ISG conserved
across many species, including mammalian species. The intronless
gene encodes an endoplasmic-reticulum-associated enzyme that
catalyzes oxidation of cholesterol to 25-hydroxycholesterol
("25HC") (Holmes et al., 2011). 25HC belongs to a diverse class of
endogenous oxysterols, the oxidation products of cholesterol. It is
widely understood as a soluble factor that control sterol
biosynthesis through regulation sterol-responsive element binding
proteins (SREBP) and nuclear receptors (Kandutsch et al., 1978;
Janowski et al., 1999). While oxysterols have unique roles in
metabolism, studies have implicated their importance in immunity.
Macrophages and B-cells express Ch25h robustly in response to
various toll-like receptor (TLR) ligands and IFN (Bauman et al.,
2009; Park and Scott, 2010). Ch25h suppresses IgA production in
B-Cells and may promote intracellular bacterial growth by induction
of prosurvival factors in macrophages (Bauman et al., 2009; Zou et
al., 2011). Like immune mediators, dysregulation of 25HC is
associated with immune pathology such as atherosclerosis (Andrew J
and Jessup, 1999), partly attributed to its induction of
inflammatory cytokine, IL-8 (Wang et al., 2012). Although these
studies support a conserved immunological role of Ch25h and 25HC,
their roles in the immune system remain elusive.
[0006] Antiviral therapies have been reported which act as viral
entry blockers; these are generally specific for particular viruses
because they block specific cellular or viral receptors required
for entry. These are exemplified, e.g., by HIV entry inhibitors
AMD3100 (see, e.g., Briz et al. J. Antimicrob. Chemother. (April
2006) 57(4): 619-62). A few broad viral entry inhibitors have been
reported which work by modification of viral membrane (see, e.g.,
Wolf et al. PNAS 2010 107 (7) 3157-3162). There is a need for new
viral inhibitors which can act generally against a broad range of
viruses.
DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1. (A) Ch25h is IFN inducible. Gene expression profile
of BMMs treated for 2.5 h with IFN.alpha. and IFN.gamma. at 62 U/mL
and 1 U/mL, respectively. Axes represent fold change in response to
IFN.alpha. or IFN.gamma. over untreated cells.
IFN.alpha.-stimulated genes that were 3-fold higher than induction
of IFN.gamma. stimulated genes were categorized as
IFN.alpha.-specific (green). Similarly, IFN.gamma. stimulated genes
were defined this way (blue). Ch25h is highlighted in red. (B)
Wildtype, IFNAR-deficient and IL-27R (TCCR/WSX-1) deficient BMMs
were stimulated with LipidA (100 ng/mL) or saline control for 4 hr
and 12 hr, respectively. CH25 expression values are presented as
RKPM values. (C) Ch25h gene expression measured by qRT-PCR of
ifnar+/+ and ifnar-/- BMMs stimulated with TLR agonists,
Pam-3-Cys(100 ng/mL), polyI:C(25 ug/mL), lipidA(10 ng/mL),
CpG-B(100 .mu.M) for 4 hours. (D) HEK293T was co-transfected
fluorescent red marker (DsRed) and with individual plasmids
encoding Tbk1, Ch25h, or vector for 36 h and infected with VSV-GFP
(0.01MOI) for 9 h. Representative contour plots are shown. (E)
Effect of overexpression of individual ISGs and Ch25h on VSV-GFP in
DsRed-positive population normalized to vector control. VSV-GFP was
quantified by the product of percent GFP-positive population and
geometric mean of the fluorescence index (MFI). Mean.+-.SEM;
*P<0.001.
[0008] FIG. 2. (A) HEK293T expressing doxycycline-inducible
construct coexpressing Ch25h-flag and red fluorescent marker
mCherry. HEK293T was transfected with vector or Ch25h encoding
plasmids for 24 h and doxycycline was added for 12 h at indicated
concentrations. Expression of Ch25h-flag was confirmed by western
blot (upper panel). After treatment, cells were infected with
VSV-GFP (0.01MOI) for 9 hrs and VSV-GFP was quantified by (% GFP+ X
GeoMean MFI). Dots represent percent positive mCherry (lower
panel). (B) RAW264.7 stably knocked down with shRNA against Ch25h
were generated by retro-viral infection. Two shRNA constructs were
made (shCh25h-A and shCH25h-B) along with scramble control.
Knockdown was confirmed by qRT-PCR. *P<0.01. (C) shCH25h-A,
shCH25hB, and scrambled stable RAW264.7 were infected with VSV-GFP
(0.1MOI) and the VSV-GFP was measured by plaque assay 14 hpi. (D)
Individual clonal population of BCR-ABL transformed B-cells from
ch25h+/+ and ch25h-/- mice were infected with VSV-GFP (0.1MOI) in
biological triplicates and the viral titers were measured by plaque
assay at indicated times. *P<0.01. (E) J2 BMM were derived from
ch25h+/+ and ch25h-/- mice and passaged for 2 weeks. The cells were
infected VSV-GFP (0.1MOI) and viral titers 14 hpi in the
supernatants was quantified by plaque assay. *P<0.01.
[0009] FIG. 3. (A) Schematic of FACs analysis of VSV-GFP in total,
DsRed- VSV-GFP was defined as % positive GFP X geometric MFI.
DsRed-positive (DsRed+) and DsRed-negative (DsRed-) populations.
(B) HEK293T transfected with DsRed and indicated expression vectors
were infected with VSV-GFP and analyzed by FACs (% positive GFP X
geometric MFI). (C) Media was collected from HEK293T after 48 h
transfection with indicated expression vector. Freshly plated
HEK293T was treated with conditioned media for 8 h and infected
with VSV-GFP (0.01MOI) for 9 h. VSV-GFP was quantified by FACs (%
positive GFP X geometric MFI). Representative histogram of FACs
data (right). *P<0.01. (D) Ifnar-/- tail derived fibroblasts
were treated with conditioned media for 12 h from HEK293T
transfected with indicated expression vector. The fibroblasts were
infected with VSV-GFP (0.1 MOI) and the viral titer in the
supernatant was measured by plaque assay. *P<0.05. (E) Ifnar-/-
derived J2 BMMs fibroblasts were treated with conditioned media for
12 h from HEK293T transfected with indicated expression vector. The
cells were infected with VSV-GFP (0.1 MOI) and the viral titer in
the supernatant was measured by plaque assay. *P<0.05.
[0010] FIG. 4. (A) CH25H converts cholesterol to
25-hydroxycholesterol (25HC, top). HEK293T was treated with
22(S)-HC, 22(R)-HC, 25HC, and the vehicle, ethanol (EtOH) for 8 h
at the indicated concentrations and infected with VSV-GFP. VSV-GFP
was quantified by FACs (% GFP+ X Geometric MFI). (B) Ch25h+/+ and
Ch25h-/- J2 BMMs were treated with 25HC(1 .mu.M) or EtOH and
infected with VSV-GFP (0.01MOI). VSV-GFP was quantified by FACs at
12 hpi. Mean.+-.SD; *P<0.02. (C) Costimulated PBMC were
pre-incubated for 24 h in conditioned media before infection with
HIV NL4-3. At 3 dpi, p24 in triplicate samples were quantified by
ELISA. Mean.+-.SEM; *P<0.001. (D) Costimulated PBMC
(1.times.10.sup.6) were pre-incubated for 24 h in 22(S)-HC (1
.mu.M), 25HC(1 .mu.M), and vehicle (EtOH) containing media before
infection with HIV NL4-3 in triplicates (30 ng of HIV strain
NL4-3). At 3 dpi, p24 was quantified by ELISA. Mean.+-.SEM;
*P<0.001. (E) HEK293T was treated with indicated conditioned
media for 12 h and infected with HSV(0.25MOI) for 24 h. HSV titer
in the supernatant was quantified by plaque assay. Mean.+-.SEM;
*P<0.001. (F) HEK293T were transfected with indicated expression
plasmids and infected with MHV68 (0.2MOI) for 24 h. MHV68 titer in
the supernatant was quantified by plaque assay. Mean.+-.SEM;
*P<0.001. (G) HeLa cells were pretreated with 25HC(1 .mu.M) or
EtOH containing media for 5 h and infected with Ebola Zaire-GFP
(EBOV) at 0.1MOI. At the indicated times, combined supernatants
from biological triplicates was measured by plaque assay. (H) HeLa
cells were pretreated with media containing indicated
concentrations of 25HC or EtOH for 12 h and infected with Nipah
virus (Bangladesh strain) at 0.1MOI. At the indicated times,
combined supernatants from biological triplicates was measured by
plaque assay. (I) HeLa cells were pretreated with media containing
indicated concentrations of 25HC or EtOH for 12 h and infected with
Russian Spring-Summer Encephalitis Virus (RSSEV) at 0.1MOI. At the
indicated times, combined supernatants from biological triplicates
was measured by plaque assay. (J) HeLa cells were pretreated with
media containing indicated concentrations of 25HC or EtOH for 12 h
and infected with Rift Valley Fever Virus ZH501 (RVFV) at 0.1MOI.
Viral titer at indicated time points was measured by plaque assay.
Values represent means of samples from triplicates. (K) HEK293T
were treated with EtOH, 22S-HC, and 25HC for 12 h and infected with
adenovirus-GFP and VSV-GFP and quantified by FACs (% GFP+ X
Geometric MFI). Mean.+-.SEM; *P<0.001.
[0011] FIG. 5. (A) HEK293 Ts were treated with conditioned media
for 12 h and infected with VSV-G pseudovirus encoding
VSV.DELTA.G-Luciferase (VSV.DELTA.G-Luc/G). The cell lysates were
collected at indicated times and measured for luciferase activity.
(B) HEK293T were treated with 25HC (5 .mu.M) at different times
relative to the VSV.DELTA.G-Luc/G infection. For time 0,
VSV.DELTA.G-Luc/G was added together to the cells for 1 h. Negative
numbers indicates addition of 25HC before infection, while positive
number indicates addition after infection. Relative Light Units
(RLU) is represented as Mean.+-.SD *P<0.01. (C) HEK293T were
treated with respective agonists for 8 h in triplicates. VSV
infection on HEK293 Ts was synchronize at 4 C, washed 3 times with
PBS, and shifted to 37 C for 30 min. VSV genomic RNA was quantified
by qRT-PCR Mean.+-.SEM; *P<0.05. (D) HEK293T were treated with
respective agonists for 8 h in triplicates. VSV infection on HEK293
Ts was synchronize at 4 C and washed 3 times with PBS. VSV genomic
RNA was quantified by qRT-PCR. (E) HEK293T was transfected with
indicated expression plasmids for 24 h and infected with
pseudovirus with encoding NipahM-beta-lactamase inside VSV-G
(VSV-G/BlaM) for 1.5 h. .beta.-lactamase activity was measured by
the cleavage of CCF2-AM dye. Response ratio is the ratio of the
cleaved form (blue 485 nm) to uncleaved (green, 525 nm) CCF2-AM.
*P<0.01, **P<0.001. (F) HEK293T was treated with indicated
conditioned media for 12 h and infected with VSV-G/BlaM.
.beta.-lactamase activity was measured by CCF2-AM response ratio.
*P<0.01, **P<0.001. (G) HEK293T was treated with indicated
concentration of 22(S)-HC, 25HC, and equivalent volume of vehicle
(EtOH) for 12 h and infected with VSV-G/BlaM. .beta.-lactamase
activity was measured by CCF2-AM response ratio. *P<0.01,
**P<0.001.
[0012] FIG. 6. (A) CEM cells were treated as indicated for 12 h and
infected with HIV-IIIB coexpressing luciferase, which can only
undergo single-round infection. Cell lysates were collected after
24 h and measured for luciferase activity. Relative Light Units
(RLU) is represented as Mean.+-.SD *P<0.05. (B) CEM cells were
treated with Integration inhibitor, elvitegravir, AMD3100(10
.mu.M), 25HC (1 .mu.M), and vehicle (EtOH) for 12 h and infected
with HIV III-B pseudovirus. At 2 and 6 hpi, total cellular DNA was
collected and HIV full-length late reverse transcript (LateRT) was
quantified by qRT-PCR with Taqman probe. (C) CEM cells were treated
with indicated conditioned media for 8 h and infected with HIV
NL4-3 encoding Vpr-BlaM (NL4-3/BlaM) in duplicates. AMD3100 serve
as positive control for entry inhibition. Beta-lactamase activity
was measured by cleavage of CCF2-AM by fluorescence plate reader.
*P<0.01. (D) CEM cells were treated with indicated 25HC(5 .mu.M)
and vehicle (EtOH) for 8 h and infected with HIV NL4-3 encoding
Vpr-BlaM (NL4-3/BlaM). AMD3100 serve as positive control for entry
inhibition. Beta-lactamase activity was measured by cleavage of
CCF2-AM. (E) Similar to FIG. 6E. CCF2-AM cleavage was confirmed by
FACs. Numbers represent percentage cells expressing cleaved form of
CCF2AM (485 nm). (F) Vero cells were transfected with Nipah F and G
receptors. 5 h after transfection, the cells were treated with
indicated conditions. The cells were fixed 21 h after transfection
and Giemsa stained. (G) Syncytias were defined by the presence of 4
or more nuclei in a common cell membrane. Relative fusion was
determined by normalizing the number of nuclei per syncytia under
the experimental conditions to the vehicle (ethanol) treated group,
set to 100%.
[0013] FIG. 7. (A) 25HC (50 mg/kg) or vehicle
(2-hydroxypropyl-.beta.-cyclodextrin) was administered 12 h before
HIV NL4-3 infection in humanized mice (DKO-hu). Treatment was
administered daily after infection. Viral titer in serum was
measured by qRT-PCR 7 dpi. Results are combined from 2 experiments.
***P<0.0001. (B) Spleens from DKO-hu mice were harvested 14 dpi
and quantified by FACs after HIV p24 intracellular staining. (C)
Percent CD4+ T-cells was compared by FACs in 25HC and EtOH treated
group. Representative FACs plots are shown (right). (D) ch25h+/+
and ch25h-/- mice were infected with MHV68-Luc (500 pfu) and the
amount of infection was quantified everyday by bioluminescence
imaging. Average total intensity from ventral, right, left, and
dorsal sides were measured for all mice. *P<0.05. (E) Average
intensity from ventral, right, left, and dorsal side of each mice
were averaged for ch25h+/+ and ch25h-/- mice. *P<0.05. MHV68
genomic DNA from ch25h+/+ and ch25h-/- infected mice 9 dpi was
quantified by qRT-PCR and normalized to a genomic promoter of ccl2
gene. *P<0.01. (F) Representative bioluminescent images of
ch25h+/+ and ch25h-/- mice 9 dpi.
[0014] FIG. 8. Indicated cell lines were treated for 8-12 h with
conditioned media from HEK293T transfected with indicated
expression vectors. They were infected with VSV at 0.01MOI for 9-14
h, depending on the cell line. VSV-GFP was quantified by FACs (%
GFP+ X Geometric MFI) and normalized to VSV-GFP in cell treated
with vector-conditioned media.
[0015] FIG. 9. (A) HEK293T was transfected with indicated
expression vector and IFN.beta.-luciferase reporter. Luciferase
activity was measured after 16 h. RLU-relative light units. (B)
IFN.beta. ELISA of conditioned media from HEK293T transfected with
indicated expression vectors after 24 h.
[0016] FIG. 10. (A) HEK293T were treated with increasing amount of
25HC and LDH values was measured after 16 h. (B) HEK293T were
treated with increasing amount of 25HC and LDH was measured after
30 and 48 h.
[0017] FIG. 11. (A) HeLa cells were pretreated with media
containing indicated concentrations of 25HC or EtOH for 12 h and
infected with RVFV (MP12 vaccine strain) at 0.1MOI. Viral titer at
indicated time points was measured by plaque assay. Values
represent means of samples from triplicates. (B) HeLa cells were
pretreated with media containing indicated concentrations of 25HC
or EtOH for 12 h and infected with Nipah virus (Bangladesh strain)
at 0.1MOI. Viral titer at indicated time points was measured by
plaque assay. Values represent means of samples from
triplicates.
[0018] FIG. 12. (A) HEK293T were treated with 25HC (2.5 uM) and
vehicle (EtOH) for 8 h and infected with VSV-GFP at 0.01 MOI. The
cells were treated against with 25HC after infection. Supernatants
were collected 24 hpi and virus was concentrated by centrifugation.
For a part of the concentrated virus, VSV genomic RNA (gRNA) was
quantified by qRT-PCR. (B) Concentrated virus from part A was
normalized based on VSV gRNA and standard plaque assay was
performed.
[0019] FIG. 13. (A) HEK293T were transfected with mature form of
SREBP1a, SREBP1c, and SREBP2 for 24 and treated with 25HC for 12 h.
The cells were infected with VSV-GFP(0.01MOI) and quantified by
FACs.
[0020] FIG. 14. (A) CEM cells were treated with indicated
conditioned media for 8 h and infected with HIV NL4-3 encoding
Vpr-BlaM (NL4-3/BlaM) in duplicates. AMD3100 serve as positive
control for entry inhibition. CCF2-AM cleavage was confirmed by
FACs. Numbers represent percentage cells expressing cleaved form of
CCF2AM (485 nm). (B) HEK293T were transfected with proviral plasmid
of HIV coexpressing GFP and treated with indicated agonists 6 h
after transfection. HIV-GFP was quantified by FACs after 48 h. (C)
Viral supernatants from part A was collected after 48 h and p24 was
quantified by ELISA.
[0021] FIG. 15. (A) Indicated cell lines were treated for 8-12 h
with conditioned media from HEK293T transfected with indicated
expression vectors. They were infected with VSV at 0.01MOI for 9-14
h, depending on the cell line. VSV-GFP was quantified by FACs (%
GFP+ X Geometric MFI) and normalized to VSV-GFP in cell treated
with vector-conditioned media. Mean.+-.SEM. (B) HEK293T was
transfected with indicated expression vector and
IFN.beta.-luciferase reporter. Luciferase activity was measured
after 16 h. RLU-relative light units. Mean.+-.SEM (C) IFN.beta.
ELISA of conditioned media from HEK293T transfected with indicated
expression vectors after 24 h.
[0022] FIG. 16. (A) HEK293T were treated with increasing amount of
25HC and LDH values was measured after 16 h. Mean.+-.SD. (B)
HEK293T were treated with increasing amount of 25HC and LDH was
measured after 30 and 48 h. Mean.+-.SD. (C) HEK293T was treated
with indicated conditioned media for 16 h. Cell viability was
measured by quantitation of ATP present in the cell by luminescent
substrate. Mean.+-.SD. (D) HeLa cells were pretreated with media
containing indicated concentrations of 25HC or EtOH for 18 h and
infected with RVFV (MP12 vaccine strain) at 0.1MOI. Viral titer at
indicated time points was measured by plaque assay. Values
represent means of samples from triplicates. (E) HeLa cells were
pretreated with media containing indicated concentrations of 25HC
or EtOH for 18 h and infected with Nipah virus (Bangladesh strain)
at 0.1MOI. Viral titer at indicated time points was measured by
plaque assay. Values represent means of samples from
triplicates.
[0023] FIG. 17. (A) HEK293T were treated with 25HC (2.5 .mu.M) and
vehicle (EtOH) for 8 h and infected with VSV-GFP at 0.01 MOI. The
cells were treated against with 25HC after infection. Supernatants
were collected 24 hpi and virus was concentrated by centrifugation.
For a part of the concentrated virus, VSV genomic RNA (gRNA) was
quantified by qRT-PCR. Mean.+-.SEM. (B) Concentrated virus from
part A was normalized based on VSV gRNA and standard plaque assay
was performed. Mean.+-.SEM. (C) HEK293T were transfected with
mature form of Srebp1a, Srebp1c, and Srebp2 for 24 and treated with
25HC for 12 h. The cells were infected with VSV-GFP(0.01MOI) and
quantified by FACs. Mean.+-.SEM. (D) Schematic of sterol synthesis
and isopentyl-PP pathway. 25HC inhibit several enzymes within the
pathway (purple): HMG-CoA Reductase (HMGCR1),
3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1), sterol-C4-methyl
oxidase (SC4MOL), squalene epoxidase (SQLE), acetyl-CoA
acetyltransferase (ACAT), farnesyl-diphosphate farnesyltransferase
(FDFT), isopentenyl-diphosphate isomerase (IDI1). Two inhibitors of
prenylation, FTI-276 and GGTI-298, are also shown. (E) HEK293 Ts
were treated with 25HC, mevalonic acid (300 uM), or both for 12 h
and infected with VSV-GFP (0.01MOI). VSV-GFP was quantified by FACs
at 9 hpi. Mean.+-.SEM. (F) HEK293T was treated as indicated for 12
h and infected with VSV-GFP (0.01MOI). VSV-GFP was quantified by
FACs at 9 hpi. (G) Cytotoxicity of treatments in part. C were
measured by ATP content and normalized to respective control (DMSO
for GGTI-298 and FTI-276, EtOH for 25HC). Mean.+-.SEM, *P<0.05.
(H) HEK293T were treated with FTI-276 at indicated concentration
and respective concentrations for 12 h and infected with
VSV-G-13laM. 13-lactamase activity was measured by blue:green ratio
of the cleaved CCF2-AM. Mean.+-.SEM. (I) HEK293T were treated with
GGTI-298 at indicated concentration and respective concentrations
for 12 h and infected with VSV-G-13laM. 13-lactamase activity was
measured by blue:green ratio of the cleaved CCF2-AM.
Mean.+-.SEM.
[0024] FIG. 18. (A) CEM cells were treated with indicated
conditioned media for 8 h and spin-infected with HIV NL4-3 encoding
Vpr-BlaM (NL4-3/BlaM) in duplicates for 2 h. CCF2-AM substrate was
added and .beta.la activity was measured for 4 h at room
temperature as shown in FIG. 6.C. After the kinetic read, CCF2-AM
cleavage was confirmed by FACs with gating on percentage cells
expressing cleaved form of CCF2AM (blue, 485 nm). (B) HEK293T were
transfected with proviral plasmid of HIV coexpressing GFP and
treated with indicated agonists 6 h after transfection. HIV-GFP was
quantified by FACs after 48 h. Mean.+-.SEM. (C) Viral supernatants
from part A were collected after 48 h and p24 was quantified by
ELISA. Mean.+-.SD.
DESCRIPTION
[0025] The present inventors have found that 25-hydroxycholesterol
(25HC) and its derivatives or analogs are useful for antiviral
therapy against a broad spectrum of enveloped viruses, both for
treatment and for prophylaxis of viral infections. Without wishing
to be bound by any particular mechanism, it is suggested that 25HC
inhibits viral entry into cells by modification of cellular
membranes. 25 HC is disclosed herein to be effective, both in vitro
and in vivo (in a subject) for inhibiting, e.g., vesicular
stomatitis virus (VSV), herpes simplex virus (HSV), murine
gammaherpes virus (MHV68), hepatitis C virus (HCV),
human-immunodeficiency virus (HIV), Ebola virus (EBOV), Rift Valley
Fever virus (RVFV), Russian Spring-Summer Encephilitis virus
(RSSEV) and Nipah viruses. Some of these viruses, such as Ebola and
Rift Valley Fever virus, are highly pathogenic.
[0026] The Examples herein demonstrate the inhibitory effect of
25HC on a variety of enveloped viruses, including highly pathogenic
viruses; and they show that 25HC reduces HIV replication in
humanized mouse models.
[0027] One advantage of the compounds and methods of the present
invention is that the compounds broadly inhibit viral infection,
including the viruses noted above and other enveloped viruses.
Furthermore, 25HC is an endogenously produced product. Hence, its
toxicity is better tolerated than other chemical compounds, such as
agents targeted against cellular metabolic functions.
[0028] One aspect of the invention is a method for inhibiting the
growth and/or proliferation and/or infectivity of a virus in a
cell, comprising administering, or causing to be administered, to
the cell, 25-hydroxycholesterol (25HC) in an amount sufficient to
inhibit the growth and/or proliferation and/or infectivity of the
virus in the cell,
[0029] wherein, if the cell is in vitro, the 25HC is administered
to the cell, and the virus is vesicular stomatitis virus (VSV),
herpes simplex virus (HSV), murine gammaherpes virus (MHV68),
hepatitis C virus (HCV), Ebola virus (EBOV), or Nipah virus;
and
[0030] wherein, if the cell is in a subject, the 25HC is
administered or caused to be administered to the subject, and the
virus is vesicular stomatitis virus (VSV), herpes simplex virus
(HSV), murine gammaherpes virus (MHV68), hepatitis C virus (HCV),
human-immunodeficiency virus (HIV), Ebola virus (EBOV), or Nipah
virus.
[0031] Embodiments of this method include a method for preventing
the viral infection of a cell (e.g., a mammalian cell) in vitro or
in a subject; and a method for inhibiting entry of the virus into a
cell (e.g., a mammalian cell) in vitro or in a subject.
[0032] In embodiments in which the 25HC is administered to a
subject, it can be administered by a route selected from the group
consisting of topical administration, oral administration, nasal
administration, rectal administration, vaginal administration,
intraperitoneal injection, intravascular injection, subcutaneous
injection, transcutaneous administration, inhalation
administration, and intramuscular injection. It can be administered
topically, vaginally, rectally, or to the buccal cavity. It can be
administered to a mucosal surface.
[0033] In embodiments in which the 25HC is administered to a
subject, it can be formulated as a cream, gel, or foam for rectal
delivery or vaginal delivery or topical administration; as a
mouthwash for delivery to the buccal cavity; or for oral or
intravenous delivery (e.g., the 25HC is solubilized in
(2-hydroxy)-beta-cyclodextrin).
[0034] In embodiments of the invention, the 25HC is administered to
a mammalian cell, and/or to a mammal, wherein the cell or mammal is
either a non-human mammal or a human.
[0035] In one embodiment of the invention, the subject (e.g. human)
to which the 25HC is administered is identified as being at risk
for an infection by the virus. In this embodiment, the 25HC is
administered prior to viral infection and prevents the viral
infection (e.g., prevent entry of the virus into cells in the
subject).
[0036] In another embodiment of the invention, the subject (e.g.
human) to which the 25HC is administered is identified as having an
infection by the virus. In this embodiment, the administration of
the 25HC treats the viral infection.
[0037] Another aspect of the invention is a method for identifying
putative inhibitors of viral entry into cells which exhibit lower
levels of side effects than does 25HC, comprising testing analogs
of 25HC in vitro for their ability to: a) exhibit anti-viral
activity; b) exhibit lower levels of cell cytoxicity (statistically
significant reductions in the level of cytotoxicity being measured
in an assay) than does a suitable control, such as 25HC; and c)
inhibit lipid metabolism to a lower level (exhibit a statistically
significantly lower level of inhibition) than does a suitable
control, such as 25HC. In embodiments of the invention, one can
employ basic metabolic assays, such as for cholesterol or
triglycerides, or can assay for SREBP processing. For example, if
an agent inhibits SREBP processing to a lower level (exhibit a
statistically significantly lower level) than does a suitable
control, such as 25HC, it is a good candidate for an agent that
does not cause undesirable side effects.
[0038] Any of a variety of enveloped viruses can be inhibited by a
method of the invention. These include, e.g., VSV, HSV, MHV68, HCV,
HIV (any of a variety of strains, which will be evident to one of
skill in the art), EBOV, RVFV, RSSEV and Nipah virus. Other
enveloped viruses that can be inhibited include, e.g., other herpes
viruses, Pox virus, Reo virus, Filo virus, Hepatitis D virus,
Corona virus, Toga virus, and other Retroviruses.
[0039] Viruses for which the inventive method can be used include
the following:
DNA viruses
[0040] Herpesviruses, including HHV-1 to HHV-8
[0041] Poxviruses, including Orthopox (smallpox virus (variola),
vaccinia virus, cowpox virus, monkeypox virus); Parapox (orf virus,
pseudocowpox, bovine papular stomatitis virus); Yatapox (tanapox
virus, yaba monkey tumor virus); Molluscipox (molluscum contagiosum
virus (MCV))
[0042] Hepadnaviruses, including Hepatitis B
RNA viruses
[0043] Flavivirus, including West Nile virus, dengue virus,
tick-borne encephalitis virus, yellow fever virus
[0044] Togavirus, including Genus Alphavirus: Sindbis virus,
Eastern equine encephalitis virus, Western equine encephalitis
virus, Venezuelan equine encephalitis virus, Ross River virus,
O'nyong'nyong virus, Chikungunya, Semliki Forest virus; and Genus
Rubivirus: Rubella virus
[0045] Coronavirus
[0046] Hepatitis C and Hepatitis D
[0047] Orthomyxovirus, including Influenzavirus A, Influenzavirus
B, Influenzavirus C, Isavirus, Thogotovirus
[0048] Paramyxovirus, including mumps, measles, respiratory
syncytial virus (RSV), parainfluenza viruses, Human
metapneumovirus, canine distemper virus (dogs), phocine distemper
virus (seals), cetacean morbillivirus (dolphins and porpoises),
Newcastle disease virus (birds), and rinderpest virus (cattle),
henipaviruses including Hendra virus (HeV) and Nipah virus
(NiV)
[0049] Rhabdovirus, including RaV (Rabies virus), VSV (Vesicular
stomatitis virus)
[0050] Bunyavirus, including Hantavirus (Hantaan virus), Nairovirus
(Dugbe virus), Orthobunyavirus (Bunyamwera virus), Phlebovirus
(Rift Valley fever virus)
[0051] Filovirus, including Cuevavirus, Ebolavirus, and
Marburgvirus
[0052] Retroviruses, including Alpharetrovirus (Avian leukosis
virus, Rous sarcoma virus), Betaretrovirus (Mouse mammary tumour
virus), Gammaretrovirus (Murine leukemia virus, Feline leukemia
virus), Deltaretrovirus (Bovine leukemia virus, Human
T-lymphotropic virus), Epsilonretrovirus (Walleye dermal sarcoma
virus), Lentivirus (Human immunodeficiency virus, Simian, Feline
immunodeficiency viruses), Spumavirus (Simian foamy virus).
[0053] When 25HC is referred to herein, it is to be understood that
this term can include stereoisomers thereof, including
diastereomers, racemates, enantiomers and other isomers of the
compound. Conventional pharmaceutically acceptable salts or
solvates of 25HC are also included.
[0054] The meanings of some of terms, as used herein, are indicated
below.
[0055] A "derivative" of a compound, as used herein, refers to a
chemically modified compound wherein the chemical modification
takes place at one or more functional groups of the compound. The
derivative however, is expected to retain, or enhance, the
pharmacological activity of the compound from which it is
derived.
[0056] As used herein, "administering" refers to local and/or
systemic administration, e.g., including enteral, parenteral,
pulmonary, and topical/transdermal administration. Routes of
administration that find use in the methods described herein
include, e.g., oral (per os (p.o.)) administration, nasal or
inhalation administration, administration as a suppository, topical
contact, transdermal delivery (e.g., via a transdermal patch),
intrathecal (IT) administration, intravenous ("iv") administration,
intraperitoneal ("ip") administration, intramuscular ("im")
administration, intralesional administration, or subcutaneous
("sc") administration, or the implantation of a slow-release device
e.g., a mini-osmotic pump, a depot formulation, etc., to a subject.
Administration can be by any route including parenteral and
transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal).
Parenteral administration includes, e.g., intravenous,
intramuscular, intra-arterial, intradermal, subcutaneous,
intraperitoneal, intraventricular, ionophoretic and intracranial.
Other modes of delivery include, but are not limited to, the use of
liposomal formulations, intravenous infusion, topical
administration, transdermal patches, etc. 25HC is effective as a
topical agent to prevent viral infections in which viruses enter
cells by penetration of the skin or transmittal across mucosal
surfaces. Such infections include, e.g., Herpes Simplex Infections,
HIV, or other sexually-transmitted diseases.
[0057] The terms "systemic administration" and "systemically
administered" refer to a method of administering the agent(s)
described herein or composition to an animal (e.g. mammal) or plant
so that the agent(s) or composition is delivered to sites in the
body, including the targeted site of pharmaceutical action, via the
circulatory system. Systemic administration to animals such as
mammals includes, but is not limited to, oral, intranasal, rectal
and parenteral (e.g., other than through the alimentary tract, such
as intramuscular, intravenous, intra-arterial, transdermal and
subcutaneous) administration.
[0058] The term "effective amount" or "pharmaceutically effective
amount" refer to the amount and/or dosage, and/or dosage regime, of
one or more agent(s) sufficient to bring about a measurable or
detectable amount of the desired result e.g., prophylaxis or
treatment of a viral infection in a subject (e.g. mammal), or
lessening the severity or delaying the progression of a viral
infection in a subject (e.g. mammal).
[0059] In general, 25HC stimulates a therapeutic response (e g,
inhibits growth and/or proliferation and/or infectivity of a virus
in a cell, and/or prevents a viral infection of a cell, and/or
which inhibits entry of a virus into a cell) to a statistically
significant degree compared to a suitable control, such as
treatment with a buffer or other solution lacking 25HC. For
example, in embodiments of the invention, 25HC can stimulate a
therapeutic response, as measured by any of a variety of
conventional assays, by about 1%, 5%, 10%, 20%, 30%, 40%, 50% 150%,
200%, 400% or 600% or more of that in an untreated control sample.
Intermediate values in these ranges are also included.
[0060] One skilled in the art can routinely determine the
appropriate dose, schedule, and method of administration for the
exact formulation of the composition being used, in order to
achieve the desired effective amount or effective concentration of
the agent in the individual patient. One skilled in the art also
can readily determine and use an appropriate indicator of the
"effective concentration" of a compound, for example, 25HC, by a
direct or indirect analysis of appropriate patient samples (e.g.,
blood and/or tissues), in addition to analyzing the appropriate
clinical symptoms of the disease, disorder, or condition.
[0061] The exact dose of 25HC or composition thereof administered
to an animal, such as a human, in the context of the present
invention will vary from subject to subject, depending on the
species, age, weight and general condition of the subject, the
severity or mechanism of any disorder being treated, the particular
agent or vehicle used, its mode of administration, other
medications the patient is taking and other factors normally
considered by an attending physician, when determining an
individual regimen and dose level appropriate for a particular
patient, and the like. The dose used to achieve a desired
concentration in vivo will be determined by the potency of the form
of the 25HC, the pharmacodynamics associated with the 25HC in the
host, with or without additional agents, the severity of the
disease state of infected individuals, as well as, in the case of
systemic administration, the body weight and age of the individual.
The size of the dose may also be determined by the existence of any
adverse side effects that may accompany the particular agent, or
composition thereof, employed. It is generally desirable, whenever
possible, to keep adverse side effects to a minimum.
[0062] For example, a dose can be administered in the range of from
about 5 ng (nanograms) to about 1000 mg (milligrams), or from about
100 ng to about 600 mg, or from about 1 mg to about 500 mg, or from
about 20 mg to about 400 mg. For example, the dose can be selected
to achieve a dose to body weight ratio of from about 0.0001 mg/kg
to about 1500 mg/kg, or from about 1 mg/kg to about 1000 mg/kg, or
from about 5 mg/kg to about 150 mg/kg, or from about 20 mg/kg to
about 100 mg/kg. For example, a dosage unit can be in the range of
from about 1 ng to about 5000 mg, or from about 5 ng to about 1000
mg, or from about 100 ng to about 600 mg, or from about 1 mg to
about 500 mg, or from about 20 mg to about 400 mg, or from about 40
mg to about 200 mg of 25HC or a composition comprising 25HC.
[0063] The phrase "cause to be administered" refers to the actions
taken by a medical professional (e.g., a physician), or a person
controlling medical care of a subject, that control and/or permit
the administration of the agent(s) at issue to the subject. Causing
to be administered can involve diagnosis and/or determination of an
appropriate therapeutic or prophylactic regimen, and/or prescribing
particular agent(s) for a subject. Such prescribing can include,
for example, drafting a prescription form, annotating a medical
record, and the like.
[0064] As used herein, the terms "treating" and "treatment" refer
to delaying the onset of, retarding or reversing the progress of,
reducing the severity of, or alleviating or preventing either the
disease or condition to which the term applies, or one or more
symptoms of such disease or condition.
[0065] The terms "subject," "individual," and "patient"
interchangeably can refer to any organism which contains
cholesterol-25-hydroxylase and thus can be treated with 25HC.
Suitable subjects include plants (e.g., rice (Oryza saliva) or
thale cress (Arabidopsis thaliana)) or animals, such as, e.g.,
poultry, the worm Caenorhabditis, or a mammal. The type of subject
being discussed herein will be evident from the context of the
discussion. In many embodiments of the invention, the subject is a
mammal, e.g. a human or a non-human primate, but also domesticated
mammals (e.g., canine or feline), laboratory mammals (e.g., mouse,
rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g.,
equine, bovine, porcine, ovine). In various embodiments, the
subject can be a human (e.g., adult male, adult female, adolescent
male, adolescent female, male child, female child) under the care
of a physician or other healthworker in a hospital, psychiatric
care facility, as an outpatient, or other clinical context. In
certain embodiments the subject may not be under the care or
prescription of a physician or other healthworker.
[0066] The term "formulation" or "drug formulation" or "dosage
form" or "pharmaceutical formulation" as used herein refers to a
composition containing at least one therapeutic agent or medication
for delivery to a subject. In certain embodiments the dosage form
comprises a given "formulation" or "drug formulation" and may be
administered to a patient in the form of a lozenge, pill, tablet,
capsule, suppository, membrane, strip, liquid, patch, film, gel,
foam, spray, or other form.
[0067] In aspects of the invention, the 25HC is in the form of a
pharmaceutical composition comprising 25 HC and a pharmaceutically
acceptable carrier. By a "pharmaceutically acceptable carrier" is
meant a material that is not biologically or otherwise undesirable,
i.e., the material may be administered to a subject without causing
any undesirable biological effects or interacting in a deleterious
manner with any of the other components of the pharmaceutical
composition in which it is contained. The carrier is naturally
selected to minimize any degradation of the active ingredient and
to minimize any adverse side effects in the subject, as would be
well known to one of skill in the art. For a discussion of
pharmaceutically acceptable carriers and other components of
pharmaceutical compositions, see, e.g., Remington's Pharmaceutical
Sciences, 18th ed., Mack Publishing Company, 1990. Some suitable
pharmaceutical carriers will be evident to a skilled worker and
include, e.g., water (including sterile and/or deionized water),
suitable buffers (such as PBS), physiological saline, cell culture
medium (such as DMEM), artificial cerebral spinal fluid, or the
like.
[0068] The term "mucosal membrane" refers generally to any of the
mucus-coated biological membranes in the body. In certain
embodiments active agent(s) described herein can be administered
herein via any mucous membrane found in the body, including, but
not limited to buccal, perlingual, nasal, sublingual, pulmonary,
rectal, and vaginal mucosa. Absorption through the mucosal
membranes of the oral cavity and those of the gut are of interest.
Thus, peroral, buccal, sublingual, gingival and palatal absorption
are contemplated herein.
[0069] In various embodiments the 25-hydroxycholesterol can be
incorporated into different therapeutic delivery systems. For
example, it can be incorporated as creams, gels, or foams to serve
as topical treatment for viral infection or for rectal or vaginal
application (e.g. to mucosal surfaces). In certain embodiments oral
or intravenous delivery, 25-hydoxycholesterol can be solubilized,
e.g., in (2-hydroxy)-beta-cyclodextrin.
[0070] In certain embodiments the 25HC is applied primarily for
prevention of infection. In certain embodiments the compound (or
formulation thereof) is applied directly to a site of viral entry
(e.g., to the skin, to the buccal cavity, rectally, vaginally,
etc.). As a cream or a solubilized form, the 25HC can be applied
directly and prior to infection. In certain embodiments they can be
used for various oral or sexually transmitted diseases, such as HSV
and HIV.
[0071] One aspect of the invention is a screening method to
identify agents (such as derivatives, analogs or modifications of
25HC) which can inhibit the growth and/or proliferation and/or
infectivity of an enveloped virus in a cell (e.g., which can
inhibit entry of the virus into the cell). The assay takes
advantage of the findings shown herein that 25HC efficiently
inhibits entry of the viruses into the cells, yet does not appear
to negatively affect metabolic cellular functions, and thus would
be expected to elicit fewer side effects than agents which target
such metabolic functions. Putative agents are tested, using
conventional methods and/or methods described herein, for three
different parameters: 1) cell cytotoxicity; 2) anti-viral
properties; 3) inhibitory effects on SREBP processing. Lactate
dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)
2,5-diphenyl-tetrazolium bromide (MTT) assays are employed to
assess cell cytotoxicity. A wide range of primary human cells are
used, which are derived from induced pluripotent stem (iPS) cells
(e.g., provided by the PSC Scientific Core) including Neuron cells,
Astrocytes, Hepatocytes, Endothelial cells, Blood Brain Barrier
cells, Lung Epithelial cells, Macrophages and Dendritic cells.
These cell lines are treated at different concentrations of test
compounds (0 uM-20 uM) and cytotoxic effects are evaluated at 12,
24, 48 and 72 hours post treatment using LDH and MTT assays. Most
of the viral infections used in this assay method are completed
within 24 hours, but tests are nevertheless performed also at 48
and 72 hours post treatment to obtain better understanding of
potential cytotoxic effects of the test compounds. For assessing
anti-viral activities, primary human cells are infected (e.g. as
described in the Examples herein) with, e.g., RVFV, LCMV, VSV, YFV
and/or influenza viruses in the presence of different
concentrations of test compounds (e.g. 25HC analogs or
derivatives), at 0 uM-20 uM. Viral concentrations in different
samples are measured by conventional plaque assays. Furthermore,
the anti-viral effects are confirmed to be due to their ability to
inhibit viral entry, using conventional methods such as those
described herein, including VSV and Influenza .beta.-lactamase
entry assays.
[0072] Finally, the test compounds are examined for their ability
to modulate lipid metabolism. SREBP1 and 2 processing are used as
the readout, using western blotting analysis to monitor levels of
active/nuclear form of SREBPs after treatment of cells with the
test compounds (e.g. new 25HC derivatives). 25HC administration
inhibits the cleavage of SREBP1 and 2 to their mature nuclear form.
Therefore, compounds are selected which do not inhibit SREBP
processing. In embodiments of the invention, putative inhibitory
compounds are tested by validation in conventional animal
models.
[0073] In embodiments of the invention, agents that appear
promising in the in vitro assays discussed above are further tested
to toxicity in vivo. For example, toxicity as measured by liver
damage can be tested in animal models by assaying for Aspartate
Aminotransferase (AST) or alanine aminotransferase, using
conventional methods.
[0074] In the foregoing and in the following examples, all
temperatures are set forth in uncorrected degrees Celsius; and,
unless otherwise indicated, all parts and percentages are by
weight.
EXAMPLES
[0075] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
The Interferon-Inducible Cholesterol-25-Hydroxylase Broadly
Inhibits Viral Entry by Production of 25-Hydroxycholesterol
Highlights
[0076] Ch25h is an IFN-dependent gene that inhibits virus by
production of soluble endogenous antiviral oxysterol
25-hydroxycholesterol (25HC).
[0077] Ch25h and 25HC broadly inhibit viruses including VSV, HSV,
HIV, MHV68.
[0078] 25HC inhibits live, highly-pathogenic Ebola, Nipah, Russian
Spring-Summer Encephilitis, Rift-Valley Fever Viruses.
[0079] 25HC inhibits viral entry of VSV and HIV.
[0080] 25HC inhibits viral mediated membrane fusion.
[0081] ch25h-deficient cells and mice have increased susceptibility
to acute viral infections.
[0082] Administration of 25-hydroxycholesterol in vivo suppresses
HIV replication in humanized mouse model.
Abstract of Example 1
[0083] Interferons (IFN) are essential cytokine for innate immunity
against viral infection and generate the cellular antiviral state
through upregulation of interferon-stimulated genes (ISGs). We
identified Cholesterol-25 hydroxylase (Ch25h) as an antiviral ISG
and demonstrated that it broadly inhibits enveloped viruses
including VSV, HSV, HIV, and MHV68. It also inhibits replication of
acutely pathogenic EBOV, RVFV, RSSEV, and Nipah under BSL4
conditions. Functional loss of Ch25h in Ch25h-knockdown and
Ch25h-deficient cell lines led to increased susceptibility to viral
infection in vitro. 25HC inhibits VSV and HIV cellular entry by
modification of cellular membrane. We further showed that this
modification causes defect in membrane fusion between virus and
cell. In vivo, administration of 25HC in humanized mice suppressed
HIV replication and rescued T-cell depletion. Moreover,
Ch25h-knockout mice demonstrated increased susceptibility to MHV68
lytic infection. Our findings show Ch25h as a unique antiviral ISG
that generates a soluble antiviral factor and demonstrate the
therapeutic potential of membrane-modifying oxysterols as viral
entry inhibitors.
Results
[0084] Ch25h is an IFN-Dependent Gene with Antiviral Activity
[0085] In a microarray analysis of IFN.alpha. and IFN.gamma.
stimulated murine bone marrow-derived macrophages (BMMs), we found
both IFNs induced expression of Ch25h within 3 hrs (FIG. 1A). A
subsequent RNAseq analysis showed the TLR4 agonist, lipidA, induced
Ch25h expression. This induction was dependent on IFN receptor
(IFNAR) but independent of IL-27, a cytokine that mediates IFN
secondary gene expression, such as IL-10 (FIG. 1B). We further
tested different TLR agonists and found dsRNA mimetic, polyI:C
(TLR3 agonist), and lipidA induced Ch25h mRNA expression highly
whereas Pam-3-Cys (TLR2 agonist) and CpG (TLR9 agonist) induced it
less. IFNAR-deficient BMMs had abrogated Ch25h expression when
treated with these agonists showing that Ch25h expression is
IFN-dependent (FIG. 1C).
[0086] In a previous study, we sought antiviral ISGs against
vesicular stomatitis virus (VSV) in a blinded and unbiased
functional screen (Liu et al., 2012). Individual ISGs in expression
plasmids were co-transfected with red fluorescent construct (DsRed)
in HEK293T cells for 36 h and subsequently infected with VSV
coexpressing GFP (VSV-GFP) for 9 h and analyzed by FACS. Active
viral replication was measured by percentage and geometric mean
fluorescence index (% GFP+ X Geometric MFI) of GFP-positive cells
in the DsRed population. TANK-binding kinase-1 (Tbk1), which is an
activator of IFN production, was used as a positive control. The
amount of infection was normalized to cells co-transfected with
DsRed and control vector. Expression of Ch25h inhibited VSV-GFP
replication by .about.70% at 9 hpi (FIGS. 1 D and E). IFN
activators like Tbk1, Ifih1 (MdaS), and Irf1 strongly inhibited VSV
as well as the RNA exonuclease, ISG20.
[0087] To validate the antiviral effect of Ch25h, we generated a
doxycycline-inducible Ch25h-flag construct co-expressing a
fluorescent-red mCherry (Ch25h-mCherry). Doxycycline addition to
HEK293T expressing this construct increased CH25H-flag expression
(FIG. 2A top) and mCherry expression in a dose-dependent manner
(FIG. 2A, bottom). When infected with VSV-GFP, HEK293T expressing
Ch25h-mCherry and treated with doxycycline exhibited a
dose-dependent inhibition of VSV-GFP compared to vector control
(FIG. 2A, bottom). Taken together, Ch25h is sufficient to inhibit
VSV.
[0088] Loss of Function of Ch25h Leads to Susceptibility to Viral
Infections In Vitro
[0089] We sought to determine whether Ch25h might play a necessary
role in the viral infection. We generated Ch25h stable knockdown
cell lines in murine macrophage cell line RAW264.7 with two
distinct shRNA sequences against Ch25h (FIG. 2B). Both knockdown
cell lines demonstrated increased VSV replication compared to
scramble control (FIG. 2C). To further validate these results, we
derived B-Cells and macrophages from Ch25h-deficient (ch25h-/-) and
matching wild-type (ch25h+/+) mice. In our experience, VSV-GFP
could not establish infection in primary cells (unpublished).
Hence, B-cells were immortalized with BCR-ABL virus and several
stable clones were isolated. We observed about 100 fold increase in
VSV-GFP replication in 3 different Ch25h-/- B-Cell clones at 48 hpi
compared to 2 ch25h+/+ B-cell clones (FIG. 2D). In parallel, we
performed VSV infection in BMMs immortalized by J2 virus (FIG. 2E).
Similarly, ch25h-/- J2 BMMs displayed 5-fold increased
susceptibility to VSV infection compared to ch25h+/+ J2 BMMs at 14
hpi. These results show that Ch25h may be required for host
antiviral immunity.
[0090] Ch25h Produces a Soluble Antiviral Factor that is not
IFN
[0091] Based on the FACs analyses of HEK293T transfected with ISGs
in FIG. 1D, we separated our analyses to examine total,
DsRed-positive (DsRed+), and DsRed-negative (DsRed-) populations
(FIG. 3A). DsRed+ population should represent cells that highly
expressed the ISG, whereas DsRed- population should represent the
low expressing population. IFN activators such as Tbk1, Irf1, and
Ifih1 inhibited VSV-GFP expression by >95% in all populations
suggesting that the high expressers (DsRed+) confer viral
resistance to low expressers (DsRed-) (FIG. 3B). This result is
consistent with IFN-mediated induction of an antiviral response in
naive cells. In contrast, the cytoplasmic exo-nuclease ISG20 that
degrades viral RNA, only inhibited VSV in DsRed+ population, but
could not confer protection to DsRed- population. Overexpression of
Ch25h also inhibited virus in both DsRed+ and DsRed- populations
suggesting that Ch25h produced a soluble factor that could confer,
in trans, antiviral activity onto other cells.
[0092] To determine if Ch25h produced a soluble antiviral factor,
we tested whether conditioned media from cells overexpressing Ch25h
had antiviral activity. HEK293T cells were transfected with vector,
interferon activators (Tbk11, Irf1, and Ifih1) or ISGs, for 48
hours and the conditioned media was filtered and transferred onto
freshly plated HEK293T cells for 8 h before infection with VSV-GFP
(0.01MOI) for 9 h. VSV-GFP measured by FACs was significantly less
in cells treated with conditioned media from Tbk1, Irf1, Ifih1,
because they contain IFN. Compared to vector controls,
Ch25h-conditioned media caused .about.80% VSV-GFP inhibition (FIG.
3C). On the other hand, conditioned-media from Isg20-transfected
cells had no effect on VSV replication. Furthermore, we have
observed inhibition of VSV growth by Ch25h conditioned media across
several human and murine cell lines including HeLa, 3T3, BHK,
Veros, MDCK, and Huh751 (FIG. 8). These results demonstrate that
Ch25h produces a soluble antiviral factor.
[0093] IFN is well known to induce many ISGs that positively
feedback and amplify IFN itself. Since there have been no soluble
antiviral ISGs described aside from IFN, we tested whether Ch25h
can induce IFN. Ch25h conditioned media had no detectable IFN.beta.
by ELISA and did not induce an IFN-stimulated responsive element
(ISRE) luciferase reporter (FIGS. 9A and 9B). More importantly,
Ch25h-conditioned media inhibited VSV replication in both ifnar-/-
fibroblasts and J2 BMMs. On the other hand, conditioned media from
IFN activators, Irf1, Ifih1, and Rig-I, were unable to confer
antiviral activity to ifnar-/- cell lines (FIGS. 3E and 3F). Taken
together, Ch25h produces a soluble factor that is not IFN and can
confer antiviral activity independent of IFNAR.
[0094] 25-Hydroxycholesterol, the Cognate Product of Ch25h, has
Antiviral Activity
[0095] Ch25h catalyzes oxidation of cholesterol to
25-hydroxycholesterol (25HC), which is a soluble oxysterol that
modulate cellular functions in an autocrine and paracrine fashion
(FIG. 4A, top). We hypothesized that the soluble antiviral factor
generated by Ch25h is 25-hydroxycholesterol. Treatment of HEK293T
cells with 25HC for 8 h inhibited VSV-GFP expression by FACs in a
dose-dependent manner with IC.sub.50 of .about.1 uM (FIG. 4A,
bottom). Some studies have shown 25HC as a weak ligand for LXR
suggesting this nuclear receptor might play a role in the antiviral
activity of 25HC (Janowski et al., 1999). Treatment of HEK293 Ts
with 22-(R)-hydroxycholesterol (22R-HC), an oxysterol that strongly
activates LXR, however, did not confer antiviral effect and neither
did 22-(S)-hydroxycholesterol (22S-HC), an inactive ligand for LXR
(FIG. 4. A, bottom). 25HC treatment of ch25h+/+ and ch25h-/- J2
BMMs also reduced VSV replication (FIG. 4B).
[0096] We tested whether the antiviral activity of 25HC was
attributed to cellular cytotoxicity. Increasing doses up to 10
uM-10 fold higher than observed IC.sub.50--of 25HC did not increase
LDH in supernatants of cells after 16 h of treatment; LDH level
increased only after 30-40 h treatment at 40 uM of 25HC (FIGS. 10A
and 10B). Similarly, Ch25h-conditioned media did not alter cell
viability as measured by cellular ATP levels (Supp. FIG. 10C).
These data show that Ch25h-conditioned media and the effective
antiviral dose of 25HC are not cytotoxic. Therefore, these results
suggest that the antiviral activity of Ch25h is carried out through
its enzymatic product, 25HC. Its antiviral activity is not
attributed to LXR function per se and--compared to the oxysterols
tested--is specific.
[0097] Ch25h-Conditioned Media and 25HC is Broadly Antiviral
[0098] To determine the breadth of antiviral activity of Ch25h, we
tested the effect of Ch25h-conditioned media and 25HC on various
viruses. For HIV, primary peripheral blood mononuclear cells were
treated with conditioned media or oxysterol and subsequently
infected with HIV NL4-3. At 3 dpi, Ch25h- and Irf1-conditioned
media caused .about.75% reduction of HIV NL4-3 p24 expression (FIG.
4C). Similarly, 25HC (1 .mu.M) inhibited p24 expression by
.about.80% at 3 dpi compared to vehicle treatment, whereas 22S-HC
had no effect (FIG. 4D). Ch25h-conditioned media also inhibited
herpes simplex virus 1 (HSV-1) by plaque assay (FIG. 4E) and
expression of Ch25h in HEK293T also inhibited MHV68 infection by
plaque assay (FIG. 4F).
[0099] HIV, HSV-1, and MHV68 are viruses that achieve chronically
persistent infections. To determine whether Ch25h-induced 25HC can
inhibit acutely pathogenic viruses, we tested the effect of 25HC on
live Ebola (EBOV-Zaire), Nipah (Bangladesh), Russian Spring-Summer
Encephalitis Virus (RSSEV), and Rift Valley Fever Virus RVFV
(wild-type strain ZH501 and vaccine strain MP12) under BSL4
conditions. FIGS. 4G, 4H, 4I, and 4J show that 1 uM of 25HC
inhibited replication of these live viruses. 25HC also inhibited
replication of Nipah and RVFV (MP12) in a dose-dependent manner
(FIGS. 11A and 11B). In contrast, a non-enveloped virus, adenovirus
coexpressing GFP, was not affected by 25HC as measured by FACs
(FIG. 4K). Taken together, Ch25h-induced 25HC has antiviral
activity against several types of enveloped DNA and RNA viruses,
while they do not have effect on non-enveloped virus.
[0100] 25HC Inhibits VSV Entry
[0101] We took advantage of tools available for VSV and HIV to
study the mechanism of Ch25h inhibition on the viral lifecycle.
First, we utilized the pseudotyped VSV.DELTA.G-Luc reporter virus
system that has the receptor-binding G gene (VSV-G) replaced with a
luciferase reporter gene (Negrete et al., 2006). When VSV-G is
provided in trans, this pseudotyped VSV reporter virus is only
capable of single-round infections because it cannot produce its
own VSV-G. Hence, quantification of luciferase activity is
indicative of viral lifecycle processes from entry to protein
synthesis. We observed that Ch25h and Irf1 conditioned media
inhibited virus reporter gene expression at the earliest time-point
(8 hpi) we can detect luciferase activity in the infected cell
lysate (FIG. 5A), suggesting Ch25h inhibits viral replication at an
early stage.
[0102] Next, we performed a time-of-addition experiment to better
elucidate the mechanism underlying the antiviral activity of 25-HC.
HEK293T cells were treated or pre-treated with 5 .mu.M 25HC at the
indicated time points. For pretreated cells, they were infected
with VSV.DELTA.G-Luc pseudovirus for 1 h without 25HC; after
washing, cells were replaced with regular media. We also added 25HC
concurrently with infection (time 0) for 1 h or added it at 1 hpi.
Interestingly, longer pre-treatment times correlated with greater
inhibition of VSV.DELTA.G-Luc expression, compared to the ethanol
vehicle treated controls. When 25HC was added concurrently with
VSV.DELTA.G-Luc pseudovirus or 1 hpi, VSV-G mediated infection was
not significantly inhibited (FIG. 5B). These results suggest that
25HC does not inhibit VSV during infection or after infection has
taken place. Rather, it is likely that 25HC establishes an
antiviral state prior to infection.
[0103] Since these data implicate early viral lifecycle steps may
be affected, we carried out experiments to determine whether 25HC
affects binding (Weidner et al., 2010). HEK293 Ts were treated for
8 h with ethanol (EtOH), 25HC (1 .mu.M), CPZ (10 ug/mL), an
endocytosis inhibitor that would have no effect on binding. To
measure binding, VSV (1MOI) was incubated with HEK293T at 4.degree.
C. for 1 h to allow for binding but not cell entry. After washing 3
times with cold PBS, total RNA was collected and VSV genomic RNA
(gRNA) was reverse-transcribed with gRNA specific primer. 25HC and
CPZ did not inhibit binding significantly (P>0.05) (FIG.
5C).
[0104] To determine if 25HC affects efficiency of fusion, we
established a VSV-G .beta.-lactamase (Bla) entry assay based on the
ability of VSV-G to be pseudotyped onto viral-like particles made
from the Bla-Nipah virus matrix fusion protein, herein called
VSV-G/BlaM (Wolf et al., 2009). VSV-G mediated fusion will result
in cytoplasmic delivery of Bla-M; by addition of lipophilic
fluorescent CCF2-AM substrate, the .beta.-lactamase activity can be
measured by the green (525 nm) to blue (485 nm) fluorescence shift
as a result of CCF2-AM cleavage (Zlokarnik et al., 1998). Hence,
efficiency of virus-cell fusion can be measured by the increase in
the ratio of blue to green (blue:green) fluorescence, which is
reflective of the .beta.-lactamase activity associated with BlaM
that was been released into the cytoplasm after VSV-G mediated
fusion (Cavrois et al., 2002; Wolf et al., 2009). Unlike the
VSV.DELTA.G-Luc pseudotyped virus, this VSV-G/BlaM entry assay does
not require transcription and translation of viral proteins for
reporter gene expression.
[0105] HEK293T cells were transfected with several ISGs for 48
hours and infected with VSV-G/BlaM. FIG. 5D showed that Ch25h and
Irf1 reduced efficiency of VSV fusion. Compared to vector control,
BlaM activity from Ch25h- and Irf1-transfected cells proceeded at a
slower rate compared to vector-transfected cells (compare the
respective slopes for the first 45 min) and plateaued at a lower
level (compare blue:green ratio at 120 min). To a lesser extent,
Ifitm3 also reduced VSV-G/BlaM entry, consistent with published
results that showed it inhibits VSV-pseudovirus infection (Brass et
al., 2009). ISG20, a viral RNA exonuclease, had no effect on viral
entry. Ch25h-conditioned media similarly inhibited VSV-G/BlaM
entry, but with a more pronounced effect than
Irf1-conditioned-media (FIG. 5E). Finally, we also observed a
dose-dependent inhibition on VSV-G/BlaM entry with treatment of
25HC at 1, 2.5, and 5 .mu.M (FIG. 5F). These results demonstrate
that the ISG, Ch25h, and its cognate product, 25HC, modulates the
target cell membrane in a manner that inhibits efficiency of
virus-cell fusion.
[0106] Since viral entry involves interactions between both the
viral and cellular membranes, we then asked if the infectivity of
the virions are affected when produced from 25HC treated cells.
HEK293T were treated with and without 25HC (2.5 uM) for 8 h and
infected with replication-competent VSV at 0.01 MOI. After a 1 h
infection period, the cells were washed and replaced with media
containing 25HC (2.5 uM). The viral supernatants from infected
cells were collected at 24 hpi, purified, and concentrated by
ultracentrifugation through a 20% sucrose cushion, which also
removed any residual 25HC. As expected, 25HC treatment caused
>80% reduction in the amount of VSV produced compared to
vehicle-treated controls as measured by qRT-PCR for the number of
viral genome copies (FIG. 12A). To assess infectivity, we measured
the infectious titer of viruses produced from 25HC- or
vehicle-treated cells after normalizing for the amount of viral
gRNA as determined above. When the titer was quantified on Vero
cells, viruses from 25HC treated cells had equivalent plaque
forming units as viruses from vehicle-treated cells (Supp. FIG.
11B), demonstrating that while 25HC exerts its antiviral effect by
altering target cell membrane properties, this effect is not
manifested in virions produced from those cells.
[0107] 25-hydroxycholesterol is a suppressor of SREBP2, which
controls sterol biosynthesis and can alter membrane sterol
composition. Hence, we tested the hypothesis that 25HC inhibits
viral infection through suppression of SREBP2. We tested whether
overexpression of active (cleaved) form of SREBPs in HEK293T would
overcome the anti-viral effect of 25HC. 25HC inhibited VSV
infection in HEK293T overexpressing active forms of SREBP1-A,
SREBP1-B, and SREBP2 (FIG. 13). These data demonstrate that the
antiviral effect of 25HC is SREBP independent.
[0108] Ch25h and 25HC Inhibits HIV Entry
[0109] We sought to validate Ch25h and 25HC antiviral mechanism on
HIV. Unlike VSV, HIV is a retrovirus that undergoes pH-independent
cellular entry. In CEM cells, 25HC inhibited >50% luciferase
expression from single round infection of pseudovirus with HIV-IIIB
envelope on a NL4-3 backbone coexpressing luciferase
(pNL4-3.Luc.-R-E) (FIG. 6A). AZT, an inhibitor of reverse
transcription, served as positive control and inhibited expression
by .about.70%. Hence, these data also suggest 25HC inhibits viral
lifecycle prior to translation.
[0110] HIV initiates reverse transcription of its genomic RNA to
DNA immediately after entry. Hence, we examined the effect of 25HC
on the production of full-length, reverse-transcribed DNA (lateRT).
CEM cells were infected with pseudotyped HIV-IIIB and lateRT was
measured by qRT-PCR. 25HC inhibited lateRT expression >99% at 2
hpi and .about.70% at 6 hpi (FIG. 6B). The HIV entry inhibitor,
AMD3100, served as positive control. Elvitegravir inhibits HIV at
the step DNA integration into the host genome and served as
negative control because it shouldn't inhibit lateRT formation.
These results show that 25HC inhibits a stage of HIV before reverse
transcription of its genome.
[0111] We next asked whether Ch25h inhibits HIV similar to VSV at
the level of entry. We coexpressed pNL4-3 with Bla-VPR fusion gene
to produced virions containing Bla-VPR (NL4-3/Bla). CEM cells
treated with Ch25h conditioned media exhibited .about.60% reduction
in viral entry compared to vector- and Isg20-conditioned media.
AMD3100 abrogated NL4-3/Bla entry (FIG. 6C). We further confirmed
our findings by FACS analysis and observed .about.50% decrease in
the number of cells expressing cleaved CCF2-AM substrate (blue
population) in CEM treated with Ch25h conditioned media compared to
control (FIG. 14A). Treatment of CEM cells with 25HC (5 .mu.M)
caused .about.60% decrease in NL4-3/Bla blue-green ratio at
endpoint (FIG. 6D) and >85% reduction in cells expressing
cleaved CCF2-AM by FACS analysis (FIG. 6E).
[0112] Since 25HC may have diverse cellular effects, we asked
whether 25HC might affect other HIV life cycle processes such as
transcription, translation, or budding. To assess whether
transcription of HIV is inhibited, we transfected HEK293 Ts with
pNL4-3 co-expressing GFP (NL4-3-GFP). Addition of 25HC 4 h post
transfection did not suppress GFP expression after 24 h suggesting
that 25HC does not affect HIV transcriptional and translational
processes (Supp. FIG. 14B). Concurrently, we measured the HIV p24
in the supernatants from HEK293T transfected with NL4-3-GFP to
assess the amount of viral budding. Compared with ethanol treated
controls, 25HC did not affect HIV p24 expression in the
supernatants of transfected cells, whereas Nelfinavir, a known
budding inhibitor, inhibited p24 expression by >50% at 24 and 48
h post transfection (FIG. 14C). Taken together, Ch25h and 25HC
inhibits efficiency of HIV membrane fusion, while 25HC treatment
does not directly affect HIV transcription, translation, and
budding processes.
[0113] 25HC Inhibits Virus-Cell Membrane Fusion
[0114] Although .beta.-lactamase data demonstrate 25HC inhibits
viral entry processes up to fusion, we sought to test whether 25HC
inhibits the viral fusion process itself. Since we have observed
25HC inhibited live Nipah replication (FIG. 4H), we sought to test
whether it would also affect its fusion process. Expression of the
Nipah fusion (F) and attachment (G) proteins by themselves induces
pH-independent cell membrane fusion and syncytia formation. Hence,
vero cells were transfected with recombinant Nipah F and G at equal
ratios for 5 h and refreshed with media containing 25HC or ethanol
(vehicle). At 21 h post transfection, cells were fixed and stained
by Giemsa. Grossly, 25HC treatment led to less syncytia formation
and fewer nuclei per syncytias compared to ethanol control (FIG.
6F). In a blinded count of numbers of nuclei per syncytia, a
standard measure of fusion, 2 uM of 25HC reduced fusion by
.about.50% and 10 uM by .about.60% relative to ethanol control
(FIG. 6G). These data demonstrate that 25HC modifies the cellular
membrane to inhibit viral membrane fusion.
[0115] 25HC Reduces HIV Infection In Vivo
[0116] To further determine the efficacy of 25HC against viral
infection in vivo, we took advantage of HIV infection in humanized
mouse model. Humanized mice were administered 25HC (50 mg/kg) 12 h
prior to infection with HIV NL4-R3A by intraperitoneal (i.p)
injection. 25HC or the vehicle, 2-hydroxypropyl-.beta.-cyclodextrin
(H.beta.CD), was administered by i.p. every day and the serum was
collected 7 dpi. Quantification HIV RNA in the serum from 2
combined experiments showed >80% reduction of HIV RNA
(copies/mL) in 25HC-treated mice compared to vehicle-treated mice
(P<0.0001) (FIG. 7A). At termination of the experiment on 14
dpi, HIV p24 was significantly lower in CD4 T-cells from spleens of
25HC treated mice than control (FIG. 7B). In CD3+ T-cell
population, which reflects live T-cells, 25HC prevented
HIV-mediated CD4 T-cell depletion compared to vehicle control in
peripheral blood leukocytes (P<0.05); this effect was less
significant in the spleen (P=0.06) (FIG. 7C). These data show that
administration of 25HC can cause antiviral effect against HIV in
vivo.
[0117] Ch25h-Deficient Mice are More Susceptible to Viral
Infections
[0118] To determine whether Ch25h has a physiological role in host
defense against viral infection, we tested whether ch25h-/- mice
had increased susceptibility to matching wild-type mice (ch25h+/+).
Since Ch25h expression inhibited MHV68 in vitro, we used MHV68
coexpressing luciferase (MHV68-Luc) to infect mice so that viral
lytic growth kinetics could be measured in real time by
bioluminescence. Eight-week old female ch25h+/+ and ch25h-/- mice
(N=4 in each group) were infected with 500 pfu of MHV68-Luc i.p.
and imaged every day after 3 dpi. Average luminescence intensities
from ventral, right, left, and dorsal side of every mouse were
measured. We observed significantly higher MHV68-Luc activity in
ch25h-/- mice over ch25h+/+ mice starting 5 dpi and maximal
difference by day 7 (FIG. 7D). MHV68-Luc activity began to wane in
both groups by 9 dpi with significantly higher activity in Ch25h-/-
mice. To validate the imaging results, Ch25h-/- spleens had
approximately .about.3.5 fold higher MHV68 genomic DNA than spleens
of Ch25h+/+ mice at 10 dpi (FIGS. 7E and 7F). These results show
that Ch25h is a physiologically important antiviral factor.
Discussion
[0119] We have identified the antiviral activity of an
IFN-inducible gene, Ch25h, through a systematic, functional screen.
Distinct from known IFN-mediated antiviral mechanisms, Ch25h
inhibits growth of a wide range of enveloped viruses by production
of a soluble oxysterol, 25-hydroxycholesterol. It also exemplifies
the only soluble antiviral ISG that is not IFN itself. Independent
of its known regulatory effect on metabolism, 25HC impairs viral
entry at the step virus-cell fusion by inducing cellular membrane
changes. In animal models, administration of 25HC reduces HIV
infection in humanized mice. Moreover, immune response against
viral infections requires Ch25h in vivo. These findings illustrate
an essential function of Ch25h in immunity.
[0120] Ifitm proteins are the only ISGs that have been described to
inhibit viral entry, after endocytosis and before primary
transcription (Brass et al., 2009; Weidner et al., 2010). The
transmembrane protein inhibits only certain viruses, suggesting it
has specific protein interactions with viral components. In
contrast, 25HC is broadly inhibitory against enveloped viruses
because it modifies host cellular membrane and perturbs the fusion
process with virus. Moreover, in our .beta.-lactamase assays,
overexpression of Ch25h inhibited VSV entry >2-fold higher than
Ifitm3 (FIG. 5D). Taken together, IFN induces these two ISGs to
block viral entry likely by disparate mechanisms.
[0121] Oxysterols have multi-faceted physiological roles. Their
permeability and solubility make them ideal rapid signaling
regulators. Many oxysterols, like 7.beta.-, 22-, 24-, 25-, and
27-hydroxycholesterol, redundantly regulate of sterol biosynthesis
through suppression SREBP2 activity (Radhakrishnan et al., 2007).
25HC also increases cellular cholesterol accessibility by directly
mobilizing cholesterol from membranes (Lange et al., 2004). While
microbial effects of some oxysterols have been appreciated in
chemistry, our study highlights the relationship of host antiviral
response and the oxysterol 25HC--illustrating that Ch25h-induced
25HC also acts as rapid, soluble viral fusion inhibitors (Moog et
al., 1998; Pezacki et al., 2009). Therefore, 25HC has multiple
functions in metabolism and in immunity.
[0122] Viruses enter cells with different types of fusion
machinery, classified structurally and biochemically. 25HC's
inhibition on virus is not specific to particular class of fusion
proteins because HIV and Ebola have class-I fusion peptides, RVFV
and RSSEV use class-II peptides, whereas VSV and HSV belong to
class III (Kielian and Rey, 2006; Vaney and Rey, 2011). It also
inhibits viruses that undergo either pH-dependent or pH-independent
fusion as exemplified by VSV and HIV, respectively. These findings
suggest that 25HC affect a more basic fusion process involving the
viral and cellular membrane. Indeed, 25HC causes membrane
expansion, increases solvent exposure of phospholipids, and
prevents the membrane condensing effect of cholesterol in bilayers
(Gale et al., 2009; Olsen et al., 2011). These changes can perturb
viral fusion, which is fundamentally dependent on membrane
properties such as spacing of lipid head groups, receptor
accessibility, membrane curvature, and fluidity (Pecheur et al.,
1998; Teissier and Pecheur, 2007). Studies on viral entry have
predominantly focused on viral fusion components and their
interactions with specific cellular receptors. How membrane
properties modulate viral fusion remains subject of further
research.
[0123] We have shown the important role of Ch25h in the context of
IFN induced antiviral state. Although 25HC have been associated
with pathological conditions like atherosclerosis and Alzheimers,
our studies suggest that it has a beneficial role in innate
immunity during viral infections. The co-morbidity association of
different chronic disease and 25HC would not warrant the
possibility of 25HC as a long-term therapeutic for viral
infections. Short-term 25HC treatment, however, suppressed HIV in
vivo and 25HC inhibited highly pathogenic Ebola and RVFV, which can
cause lethality within days.
Materials and Methods
[0124] Cells and Reagents:
[0125] RAW and HEK293T cells were obtained from ATCC and grown in
standard DMEM with 5% FBS, 1% Penicillin/Streptomycin (GIBCO). CEM
cells were cultured in RPMI media supplemented with 10% fetal calf
serum (Hyclone) and 1% Pen/Strep (Invitrogen). Dr. Glen Barber
(University of Miami, Fla.) provided VSV-GFP. MHV68-Luc was
provided by Dr. Ren Sun in MIMG in UCLA. Luciferase activity was
measured using firefly luciferase substrate kit (Promega). LDH
Assay and ATP cell viability (Promega) were done on cells treated
with Ch25h-conditioned media and 25HC according to manufacturer's
instructions.
[0126] To make bone marrow derived macrophages (BMMs), bone marrow
was harvested from 6-8 week C57B/L6 mice (Jackson Labs) and
differentiated with 10 ng/mL of M-CSF in DMEM+ 10% FBS for 7 days.
On day 6 the media was replaced and on day 7 the cells were
stimulated with IFN.alpha. or IFN.gamma. (PBL Interferon Source).
The cells were treated for 2.5 hours and harvested in Trizol
(Invitrogen). The RNA was isolated by isopropanol precipitation for
microarray analyses. For J2 immortalized macrophages, bone marrow
was infected with J2 retrovirus. A retrovirus expressing v-raf and
c-myc expressing cell line was established (called GG2EE) and grown
in RPMI (10 mM Hepes ph7.8, 10% FBS, 1% Pen/strep). Virus
containing supernatant was harvested and filtered through 0.45
.mu.M filter (Palleroni et al., 1991). For BCR-ABL transformed
B-cells were derived by infecting bone marrow with BCR-ABL
retrovirus as described previously (Scherle et al., 1990). Stable
knockdown in RAW264.7 were generated using pSiren shRNA knockdown
system (Clonetec) according to the published protocol. Knockdown
primers sequences are available by request. Tail-derived
fibroblasts were derived by skinning the tails of mice and
incubating them directly in culture dishes in DMEM 10% FBS. Cells
were scraped and re-plated after 7 days.
[0127] Expression plasmids were obtained from Genecopoeia, Inc.
Doxycycline inducible expression system was purchased from
Clonetec. SREBP2 expression plasmids were gifts from Dr. Elizabeth
Tarling and Dr. Peter Edwards (UCLA). SREBP1 expression plasmids
were gifts from Dr. Steven Bensinger (UCLA).
VSV, HSV, and MHV68 Viral Plaque Assay
[0128] HEK293T and RAW264.7 were infected with VSV-GFP at 0.01 MOI
for 1 h and the media was changed with fresh media. For J2 BMMs and
BCR-ABL B-cells, 1MOI VSV-GFP was used. Approximately 150 uL of
supernatants were collected at various timepoints between 8-16 hpi
for plaque assay. For HSV and MHV68, 0.25MOI was used for infection
and supernatants were collected at 24 hpi.
[0129] Plaque Assays were done on Vero cells in 12-well plates at
2.times.10.sup.5 and 2.times.10.sup.4 cells per well for VSV and
MHV68 plaque assay, respectively. Supernatants from infected cells
were serially diluted and infected on Veros for 1 hr. The cells
were then covered with growth medium containing 0.6% low-melting
point agarose. Plaques stained with crystal violet 0.5% (m/v) in
20% ethanol (v/v) and were counted after 16 hrs or 6 days for VSV
and MHV68, respectively.
[0130] VSV Screening and Flow-Cytometry:
[0131] HEK293T cells were plated 12 wells on collagen coated plates
0.5 mg/mL rat-tail collagen I (BD Biosciences) in PBS. Individual
ISGs expression plasmid was transfected with DsRed construct
(Clontech) at 3:1 ratio Fugene 6 (Roche) transfection reagents.
After 36 hours, the cells were infected as described above. At 9
hpi, cells were collected in 2% paraformaldehyde solution in PBS.
FACs was done with standard compensation (FACSCaliber,
BDBiosciences) and the data was analyzed using CellQuest
(BDBiosciences).
[0132] Ebola, RSSEV, and RVFV Infections
[0133] HeLa cells were pre-treated with 25HC (1 .mu.M) or EtOH
containing medium for 5 hrs prior infection with Ebola-Zaire-GFP
(EBOV) or Rift Valley fever virus (RVFV) wild type strain ZH501
(MOI 0.1), respectively. Cell culture supernatant aliquots were
harvested at the indicated time points and pooled from biological
triplicates, prior virus titration by plaque assay. For infection
with RVFV vaccine strain MP-12 or Russian Spring-Summer
encephalitis virus Sofjin strain (RSSEV), HeLa cells were
pre-treated with 25HC (1 .mu.M) or EtOH containing medium for 18
hrs prior infection. Plaque Assays were performed on Vero cells
(for EBOV, and RVFV) in 12-well plates or BHK-SA cells (for RSSEV)
in 6 well plates. Cells were infected for 1 hr at 37.degree. C.
with serial 10-fold dilutions of supernatant aliquots from infected
cells. The cells were then overlain with growth medium containing
0.6% methyl cellulose (for EBOV and RVFV) or 0.5% tragacanth gum
(for RSSEV). After 3 days (RVFV), 4 days (RSSEV) and 10 days
(EBOV), cells were fixed with 10% buffered formalin, stained with
crystal violet and plaques counted. All work involving EBOV, RSSEV
and wild-type RVFV, were performed at the Robert E. Shope BSL-4
laboratory at UTMB.
[0134] HIV Infections in hPBMCs
[0135] Human peripheral blood mononuclear cells (PBMC) were
obtained from the UCLA Virology Core. These cells were cultured in
RPMI Medium 1640 (Invitrogen) containing 10% FBS, 100 units/ml of
Penicillin+100 .mu.g/ml of Streptomycin (Pen/Strep, Invitrogen),
and 20 units/ml of interleukin-2 (Roche). PBMC were costimulated
for 3 days with plate-bound anti-CD3 and soluble anti-CD28
antibodies as previously described (Korin and Zack, 1999).
Costimulated PBMC were pre-incubated for 24 h at a density of
3.times.10.sup.6 cells/ml in conditioned media before infection
with HIV. Infections were set-up in 200 .mu.l volumes of
conditioned media containing 10.sup.6 cells, 30 ng of HIV strain
NL4-3 (as determined by p24 antigen ELISA [Beckman Coulter]), and
10 .mu.g/ml of polybrene. The mixture was incubated for 2 h at
37.degree. C. on a rocking platform. Following infection, cells
were washed twice with media and then split into triplicate 2 ml
cultures in conditioned media, each containing 3.3.times.10.sup.5
cells. At various post-infection timepoints, 100 .mu.l of cell-free
supernatant was removed and added to 2% Triton-x-100 in PBS for
storage at 4.degree. C. before quantitation of p24 concentration by
ELISA.
[0136] VSV-G Pseudotyped VSV-G Luciferase Pseudovirus
Production
[0137] VSV-G pseudotyped VSV-G luciferase pseudo-virus
(VSV.DELTA.G-Luc/G) was generated by methods previously described
(Takada et al., 1997) and concentrated by ultracentifugation on 20%
sucrose cusion. The VLPs were resuspended in NTE buffer (100 mM
NaCl; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA) and stored in -80 C. The
concentrations used to generate linear range of luciferase signal
were determined empirically.
[0138] VSV-G/BlaM Production
[0139] A previously described construct encoding NipahM1 fusion
with beta-lactamase (BlaM) was used to package inside VSV-G (Wolf
et al., 2009). HEK293 Ts were transfected with constructs encoding
BlaM and VSV-G or BlaM alone (bald) at 3:1 ratio in 10 cm dishes by
PEI transfection reagent. The viral supernatants were collected,
clarified, and concentrated by ultracentrifugation at >75,000 g
on 20% sucrose cushion,
[0140] HIV IIIB Pseudotyped Virus Production
[0141] Envelopes and backbone were obtained through the NIH AIDS
and Research and Reference Reagent Program. Pseudovirons were
generated by cotransfection of 293T cells with envelope deleted
LucRE-vector and envelope expressing vector at a 3:1 .mu.g ratio
with Bioline Bio T transfection reagent. 72 hours post transfection
viral supernatant was collected, clarified by low speed
centrifugation and stored at -80 c. The number of infectious virus
particles was determined by serial dilution assay on Ghost HI-X4
cells, cells that express GFP controlled by a HIV LTR promoter.
Briefly, 4.times.10.sup.4 Ghost HI-X4 cells were seeded into a 48
well dish. 24 hour later, cells are infected with 2 fold serially
diluted psuedovirons. 48 hours later, cells were collected and
percent positive cells were determined using flow cytometry.
[0142] CEM Infection with Pseudotyped IIIB Virus
[0143] CEMs cells were treated with 25HC, 22HC or EtOH for a
minimum of six hours. Prior to infection previously untreated cells
were incubated with AMD3100, AZT, Elvitegravir or Nelfinavir at a
concentration of 10 um or 20 um for a minimum of 15 minutes. IIIB
pseudovirus was used to infect treated and untreated CEM cells at
0.1MOI. Infections were spin inoculated for 60 minutes at 2,000
RPM, at 37 C. After spin inoculation, cells were transferred into a
37 C incubator. 48 hours post infection cells were lysed with 1%
triton-X 100 and assayed for luciferase activity. P24 Assay was
done in with PerkinElmer's HIV-1 P24 Elisa kit (NEK050B).
Accompanied protocol was followed.
[0144] Generation of VPR-BlaM Fusion Gene
[0145] VPR-BlaM was generated by overlapping per and cloned into
PCDNA3.1. VPR was amplified from SG3.DELTA.. Catalytically
optimized beta lactamase described previously in (cite) was
amplified with primers (3,4). The two per products were used in the
overlapping per using primers (1,4) to generate VPR-BlaM. VPR-BlaM
per product was cloned into pcdna3.1 expression vector previously
cut with BamHI and XhoI using the Infusion system (CloneTech).
[0146] NL4-3 VPR-BlaM Virus Production
[0147] NL4-3 VPR-BlaM was produced in according to the methods
outlined previously (Cavrois et al., 2002). Briefly, 2 ug of VPR
BlaM, 1 ug of padvantage (Promega) and 8 ug of NL4-3 were
cotransfected into 293 Ts in 10 cm plate with PEI reagent. 48 hours
post transfection viral supernatant was collected, clarified by low
speed centrifugation, and concentrated on 20% sucrose cushion. VLPs
were resuspended in small volume of NTE and stored at -80.degree.
C.
[0148] NL4-3 VPR-BlaM Infection of CEM Cells
[0149] CEMs cells were treated with 25HC or EtOH for a minimum of
six hours. Prior to infection previously untreated cells were
incubated with AMD3100 at a concentration of 20 um for a minimum of
15 minutes. Concentrated NL4-3 BlaM or Bald virus was added to
treated CEM cells. Infections were spin inoculated for 60 minutes
at 2,000 RPM, at 37 degrees Celsius. After spin inoculation, cells
were transferred into a 37 degree incubator for 2 hours. Free
floating virus was inactivated and CEM cells were washed twice.
CCF2-AM (Invitrogen) was added according to manufacture's protocol.
Kinetic readings were taken for 1-3 hours. After the kinetic
reading the cells were washed with FACS Buffer, fixed with 2%
paraformaldehyde and examined by flow cytometry. Data was analyzed
using FlowJo (Tree Star Inc.).
[0150] Nipah Fusion Assay
[0151] Vero cells were plated in 6-well dish at 5.times.10.sup.5
per well overnight and transfected with 0.5 ug of expression
plasmids encoding Nipah F and G in OptiMEM (Invitrogen). At 5 h
post transfection, media was changed to DMEM (10% FBS) with or with
out 25HC at the indicated concentrations. The cells were fixed by
methanol 21 h after transfection for 10 min, stained with Giemsa
stain for 2 h, and decolorized with 95% ethanol. Nuclei inside
syncytia were counted under light microscopy. Syncytia were defined
as four or more nuclei within a common cell membrane. Relative
fusion was defined by normalizing the number of nuclei per syncytia
formed under the experimental conditions to that formed by in
vehicle (ethnnol) treated cells, which was set at 100%.
[0152] PCR
[0153] Cells were collected in trizol and RNA was isolated by
standard isopropanol precipitation. RNA was quantitated and 1 .mu.g
of RNA was reversed transcribed using IScript (BioRad) according to
the manufacturer's instructions with either random hexamer as
primers. Q-PCR analysis was done using the iCycler thermocycler
(Bio-Rad). Q-PCR was conducted in a final volume of 20 .mu.L
containing: Native Taq polymerase (Invitrogen), 1.times. Taq buffer
(Stratagene), 125 .mu.M dNTP, SYBR Green I (Molecular Probes), and
Fluoroscein (Bio-Rad), and 1% cDNA. Amplification conditions were:
95.degree. C. (3 min), 40 cycles of 95.degree. C. (20 s),
55.degree. C. (30 s), 72.degree. C. (20 s). Expression values were
normalized to L32 control and fold induction was normalized to
untreated control.qRT-PCR of Ch25h was done with primers: Ch25h
fwd: 5'-TGCTACAACGGTTCGGAGC-3' (SEQ ID NO:1). Ch25h rev:
5'-AGAAGCCCACGTAAGTGATGAT-3' (SEQ ID NO:2). L32 fwd:
5'-AAGCGAAACTGGCGGAAAC-3'(SEQ ID NO:3); L32 rev:
5'-TAACCGATGTTGGGCATCAG-3' (SEQ ID NO:4).
[0154] For detection of VSV genomic RNA, cells infected with VSV
was collected in Trizol and RNA was isolated and reverse
transcribed with VSV specific primer N1-5'
GATAGTACCGGAGGATTGACGACTA (SEQ ID NO:5) using Superscript II
(Invitrogen) according to manufacturer's protocol. Real time PCR
with Taqman probe with conditions described above. VSV fwd:
5'-GATAGTACCGGAGGATTGACGACTA-3' (SEQ ID NO:6); VSV rev:
5'-TCAAACCATCCGAGCCATTC-3 (SEQ ID NO:7); VSV Probe: 5'
(FAM)-TGCACCGCCACAAGGCAGAGA-(TAMRA)-3' (SEQ ID NO:8).
[0155] CEM cells were infected with HIV IIIB expressing GFP and or
Luciferase. After spinoculation, the cells were collected at
indicated time and total DNA was extracted with DNAeasy Blood
&Tissue Kit (Qiagen). Full length LateRT was measured by Taqman
qRT-PCR as described previously (Butler et al., 2001) and
normalized to mitochondria DNA. Primers used: late RT forward:
5'-TGTGTGCCCGTCTGTTGTGT-3' (SEQ ID NO:9); late RT reverse:
5'-GAGTCCTGCGTCGAGAGAGC-3' (SEQ ID NO:10); late RT probe,
5'-(FAM)-CAGTGGCGCCCGAACAGGGA-(TAMRA)-3' (SEQ ID NO:11);
Mitochondrial forward primer, 5'-ACCCACTCCCTCTTAGCCAATATT-3' (SEQ
ID NO:12); mitochondrial reverse primer, 5'-GTAGGGCTAGGCCCACCG-3'
(SEQ ID NO:13).
[0156] RNA Isolation and RNAseq of Bone Marrow Derived Macrophage
Stimulation
[0157] 5.times.10.sup.5 BMMs from wildtype (C57BL/6),
IFNAR-deficient and IL-27R (TCCR/WSX-1) deficient mice were
stimulated with Lipid A (100 ng/mL) or saline control for 4 hr and
12 hr, respectively. Cells were harvested in Trizol (Invitrogen)
and RNA was isolated via chloroform extraction.
[0158] Prior to cDNA library construction for RNA-Seq analyses, RNA
was quantified and assessed for quality (RNA Integrity Value) using
an Agilent 2100 Bioanalyzer (Santa Clara, Calif.). 1 ug of RNA per
condition was used for library construction using TruSeq SBS Kit v3
.sub.(FC-401-3001) according to the manufacturers guidelines
(Illumina, San Diego, Calif.). Multiplex Sequence Analysis was
performed using a Illumina HiSeq2000 Single End 100 bp read
parameters according to the manufacturers guidelines
(www.illumina.com). Sequencing was performed by the Southern
California Genotyping Consortium (SCGC) in the Epigenetics and
Genetics Core at UCLA. Sequence reads from each cDNA library (100
bp, single-read) were trimmed to 80 bp long and mapped onto the
mouse genome build NCBI37/mm9 using Bowtie (bowtie-0.12.1,
http://bowtie-bio.sourceforge.net/index.shtml) with setting `-v 2
-k 11 -m 10 -t-best-strata`. The mappable data were then processed
by the ERANGE v. 3.3 RNA-seq analysis program (Mortazavi et al.,
2008). Assuming total transcriptional activity is comparable
between different cell types, the obtained data (data units in
RPKM, reads per kilobase exon model per million mapped reads) were
first log.sub.2 transformed and linearly normalized between
individual samples, then averaged among biological replicates or
triplicates. At the same time, in order to find genes that were
changed in expression between two populations to a statistically
significant degree, ERANGE processed data were analyzed by the
Bioconductor DEGseq program (Wang et al., 2010)
(http://www.bioconductor.org/packages/2.6/bioc/html/DEGseq.html)
(data units in RPM, reads per million mapped reads, method="MARS,"
p<0.001). Data is presented using RPKM values.
[0159] Construction of DKO-hu Mice
[0160] DKO-hu mice were constructed as previously reported (Zhang
et al., 2006). Briefly, human CD34+ cells were isolated from 17-20
weeks old fetal liver tissues. The cell suspension released from
the liver was filtered through a 70-um cell strainer (BD Falcon)
centrifuged at 150.times.g for 5 min to get rid of hepatocytes. The
mononuclear cells were purified through Ficoll gradient (GE
Healthcare Bioscience AB). Cells were labeled with CD34 MicroBead
Kit from Miltenyi Biotec, then CD34+ cells were positive selected
with autoMACS followed the vendor's instruction (Miltenyi Biotec,
Germany). 1-4=105 CD34+ HSPC cells were injected into the liver of
each DKO mouse at 1 to 3 days old, which has been previously
irradiated at 400 rad. Transplanted mice were bled through tail
vein at 3-4 months after transplant to check human cell
reconstitution by flow cytometry. All animal experiences were
reviewed and approved by the University of North Carolina--Chapel
Hill Institutional Animal Care and Use Committee.
[0161] HIV Infection in DKO-hu Mice
[0162] We used an HIV molecular clone with a highly pathogenic dual
tropic envelope, R3A in NL4-3 Backbone for infection. HIV-1 viral
stocks were produced in 293T cells and expanded in PHA activated
PBMC, and titered on Hela-CD4-LTR-gal cells (NIH AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID). DKO-hu mice
with stable human leukocyte reconstitution were administered 50
mg/kg of 25HC or the vehicle control
(2-hydroxypropyl)-.beta.-cyclodextrin (HBCD) intraperitonial (i.p.)
injection for 12 h before infection with HIV NL4-R3A at 5 ng of
p24/mouse by intravenously injection (i.v.). Mice were administered
50 mg/kg of 25HC or HBCD control every day. DKO-hu mice infected
with mock supernatant were included as control groups. HIV
replication (genome copy/ml in the plasma) was measured by the
Roche Amplicor Monitor v.1.5 qRT-PCR assay (Roche Diagnostics
Corporation, Indianapolis, Ind.), or by p24 intracellular staining.
Intracellular staining and FACs analysis were done as previously
described (Zhang et al., 2011).
[0163] Mouse Infections and Bioluminescence Imaging
[0164] C57BL/6 and ch25h-/- mice were purchased from Jackson. Mice
were first anaesthetized by intraperitoneal (i.p.) injection with
200 mg/kg ketamine, 4 mg/kg xylazine in PBS. MHV68 (500 pfu) in 200
uL of PBS was administered by i.p. On days 3 following infection,
mice were imaged using the in vivo imaging system (IVIS, Xenogen).
Briefly, mice were anaesthetized by intraperitoneal injection with
200 mg/kg ketamine, 4 mg/kg xylazine in PBS, followed by
intraperitoneal injection of 3 mg D-luciferin/mouse prior to
imaging. Grayscale photographs and color images of imaged mice were
superimposed with LivingImage (Xenogen) and Igor (Wavemetrics)
programs, similar to that previously described. The mice were
imaged on dorsal, ventral, right, and left side until the maximal
luminescence has passed. The average and maximum photon flux value
was measured for each mouse at every angle and expressed as
photons/sec/cm.sup.2/steradian. These values were averaged for all
the mice. At 9 dpi, mice were euthanized the spleens were extracted
and homogenized in DMEM. Total DNA was extracted using DNeasy Blood
& Tissue Kit (Qiagen) and MHV68 DNA was quantified by
qRT-PCR.
[0165] In vivo analysis of 25HC-mediated inhibition of other
enveloped viruses, including viruses disclosed herein, is conducted
using conventional assays, in established animal models. The animal
models differ for various viruses being tested. For example,
suitable mouse, rat, chicken and non-human primate models are
available. It is expected that these viruses, too, will be
inhibited by 25HC, in a similar manner as shown herein for HIV.
References for Example 1
[0166] Andrew J, and Jessup, W. (1999). Oxysterols and
atherosclerosis. Atherosclerosis 142, 1-28. [0167] Bauman, D. R.,
Bitmansour, A. D., McDonald, J. G., Thompson, B. M., Liang, G., and
Russell, [0168] D. W. (2009). 25-Hydroxycholesterol secreted by
macrophages in response to Toll-like receptor activation suppresses
immunoglobulin A production. Proceedings of the National Academy of
Sciences 106, 16764-16769. [0169] Brass, A. L., Huang, I.-C.,
Benita, Y., John, S. P., Krishnan, M. N., Feeley, E. M., Ryan, B.
J., Weyer, J. L., van der Weyden, L., Fikrig, E., et al. (2009).
The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1
Virus, West Nile Virus, and Dengue Virus. Cell 139, 1243-1254.
[0170] Butler, S. L., Hansen, M. S. T., and Bushman, F. D. (2001).
A quantitative assay for HIV DNA integration in vivo. Nat Med
7,631-634. [0171] Cavrois, M., de Noronha, C., and Greene, W. C.
(2002). A sensitive and specific enzyme-based assay detecting HIV-1
virion fusion in primary T lymphocytes. Nat Biotech 20, 1151-1154.
[0172] Degols, G., Eldin, P., and Mechti, N. (June). ISG20, an
actor of the innate immune response. Biochimie 89, 831-835. [0173]
Gale, S. E., Westover, E. J., Dudley, N., Krishnan, K., Merlin, S.,
Scherrer, D. E., Han, X., Zhai, X., Brockman, H. L., Brown, R. E.,
et al. (2009). Side Chain Oxygenated Cholesterol Regulates Cellular
Cholesterol Homeostasis through Direct Sterol-Membrane
Interactions. Journal of Biological Chemistry 284, 1755-1764.
[0174] Garcia, M. A., Gil, J., Ventoso, I., Guerra, S., Domingo,
E., Rivas, C., and Esteban, M. (2006). Impact of Protein Kinase PKR
in Cell Biology: from Antiviral to Antiproliferative Action.
Microbiology and Molecular Biology Reviews 70, 1032-1060. [0175]
Holmes, R., VandeBerg, J., and Cox, L. (2011). Genomics and
proteomics of vertebrate cholesterol ester lipase (LIPA) and
cholesterol 25-hydroxylase (CH25H). 3 Biotech 1, 99-109. [0176]
Janowski, B. A., Grogan, M. J., Jones, S. A., Wisely, G. B.,
Kliewer, S. A., Corey, E. J., and Mangelsdorf, D. J. (1999).
Structural requirements of ligands for the oxysterol liver X
receptors LXR.alpha. and LXR.beta.. Proceedings of the National
Academy of Sciences 96, 266-271. [0177] Kandutsch, A., Chen, H.,
and Heiniger, H. (1978). Biological activity of some oxygenated
sterols. Science 201, 498-501. [0178] Kielian, M., and Rey, F. A.
(2006). Virus membrane-fusion proteins: more than one way to make a
hairpin. Nat Rev Micro 4, 67-76. [0179] Korin, Y. D., and Zack, J.
A. (1999). Nonproductive Human Immunodeficiency Virus Type 1
Infection in Nucleoside-Treated G0 Lymphocytes. Journal of Virology
73,6526-6532. [0180] Lange, Y., Ye, J., and Steck, T. L. (2004).
How cholesterol homeostasis is regulated by plasma membrane
cholesterol in excess of phospholipids. Proceedings of the National
Academy of Sciences of the United States of America 101,
11664-11667. [0181] Liu, S.-Y., Sanchez, D. J., Aliyari, R., Lu,
S., and Cheng, G. (2012). Systematic identification of type I and
type II interferon-induced antiviral factors. Proceedings of the
National Academy of Sciences 109, 4239-4244. [0182] Moog, C.,
Aubertin, A., Kim, A., and Luu, B. (1998). Oxysterols, but not
cholesterol, inhibit human immunodeficiency virus replication in
vitro. Antiviral Chemistry & Chemotherapy 9, 491-496. [0183]
Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., and Wold,
B. (2008). Mapping and quantifying mammalian transcriptomes by
RNA-Seq. Nat Meth 5, 621-628. [0184] Negrete, O. A., Wolf, M. C.,
Aguilar, H. C., Enterlein, S., Wang, W., Muhlberger, E., Su, S. V.,
Bertolotti-Ciarlet, A., Flick, R., and Lee, B. (2006). Two Key
Residues in EphrinB3 Are Critical for Its Use as an Alternative
Receptor for Nipah Virus. PLoS Pathog 2, e7. [0185] Olsen, B. N.,
Schlesinger, P. H., Ory, D. S., and Baker, N. A. (2011).
25-Hydroxycholesterol Increases the Availability of Cholesterol in
Phospholipid Membranes. Biophysical Journal 100, 948-956. [0186]
Palleroni, A. V., Varesio, L., Wright, R. B., and Brunda, M. J.
(1991). Tumoricidal alveolar macrophage and tumor infiltrating
macrophage cell lines. Int. J. Cancer 49, 296-302. [0187] Park, K.,
and Scott, A. L. (2010). Cholesterol 25-hydroxylase production by
dendritic cells and macrophages is regulated by type I interferons.
Journal of Leukocyte Biology 88, 1081-1087. [0188] Pecheur, E.-I.,
Sainte-Marie, J., Bienvenue, A., and Hoekstra, D. (1998). Lipid
Headgroup Spacing and Peptide Penetration, but Not Peptide
Oligomerization, Modulate Peptide-Induced Fusion.dagger..
Biochemistry 38, 364-373. [0189] Perez-Caballero, D., Zang, T.,
Ebrahimi, A., McNatt, M. W., Gregory, D. A., Johnson, M. C., and
Bieniasz, P. D. (2009). Tetherin Inhibits HIV-1 Release by Directly
Tethering Virions to Cells. Cell 139, 499-511. [0190] Pezacki, J.,
Sagan, S., Tonary, A., Rouleau, Y., Belanger, S., Supekova, L., and
Su, A. (2009). Transcriptional profiling of the effects of
25-hydroxycholesterol on human hepatocyte metabolism and the
antiviral state it conveys against the hepatitis C virus. BMC
Chemical Biology 9, 2. [0191] Radhakrishnan, A., Ikeda, Y., Kwon,
H. J., Brown, M. S., and Goldstein, J. L. (2007). Sterol-regulated
transport of SREBPs from endoplasmic reticulum to Golgi: Oxysterols
block transport by binding to Insig. Proceedings of the National
Academy of Sciences 104, 6511-6518. [0192] Scherle, P. A.,
Dorshkind, K., and Witte, O. N. (1990). Clonal lymphoid progenitor
cell lines expressing the BCR/ABL oncogene retain full
differentiative function. Proceedings of the National Academy of
Sciences 87, 1908-1912. [0193] Takada, A., Robison, C., Goto, H.,
Sanchez, A., Murti, K. G., Whitt, M. A., and Kawaoka, Y. (1997). A
system for functional analysis of Ebola virus glycoprotein.
Proceedings of the National Academy of Sciences 94, 14764-14769.
[0194] Teissier, E., and Pecheur, E.-I. (2007). Lipids as
modulators of membrane fusion mediated by viral fusion proteins.
European Biophysics Journal 36, 887-899. [0195] Vaney, M.-C., and
Rey, F. A. (2011). Class II enveloped viruses. Cellular
Microbiology 13, 1451-1459. [0196] Wang, F., Xia, W., Liu, F., Li,
J., Wang, G., and Gu, J. (2012). Interferon regulator factor
1/retinoic inducible gene I (IRF1/RIG-I) axis mediates
25-hydroxycholesterol-induced interleukin-8 production in
atherosclerosis. Cardiovascular Research 93, 190-199. [0197] Wang,
L., Feng, Z., Wang, X., Wang, X., and Zhang, X. (2010). DEGseq: an
R package for identifying differentially expressed genes from
RNA-seq data. Bioinformatics 26, 136-138. [0198] Weidner, J. M.,
Jiang, D., Pan, X.-B., Chang, J., Block, T. M., and Guo, J.-T.
(2010). Interferon-Induced Cell Membrane Proteins, IFITM3 and
Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct
Mechanisms. Journal of Virology 84, 12646-12657. [0199] Wolf, M.,
Wang, Y., Freiberg, A., Aguilar, H., Holbrook, M., and Lee, B.
(2009). A catalytically and genetically optimized
beta-lactamase-matrix based assay for sensitive, specific, and
higher throughput analysis of native henipavirus entry
characteristics. Virology Journal 6, 119. [0200] Zhang, L., Jiang,
Q., Li, G., Jeffrey, J., Kovalev, G. I., and Su, L. (2011).
Efficient infection and impairment of pDC in the bone marrow and
peripheral lymphoid organs during early HIV-1 infection in
humanized rag2-/-.gamma.C-/- mice in vivo. Blood. [0201] Zhang, L.,
Kovalev, G. I., and Su, L. (2006). HIV-1 infection and pathogenesis
in a novel humanized mouse model. Blood. [0202] Zlokarnik, G.,
Negulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L.,
Whitney, M., Roemer, K., and Tsien, R. Y. (1998). Quantitation of
Transcription and Clonal Selection of Single Living Cells with
.beta.-Lactamase as Reporter. Science 279, 84-88. [0203] Zou, T.,
Garifulin, O., Berland, R., and Boyartchuk, V. L. (2011). Listeria
monocytogenes Infection Induces Prosurvival Metabolic Signaling in
Macrophages. Infection and Immunity 79, 1526-1535.
Example 2
Supplemental Information
[0204] The data shown in Example 1 are extended as shown in FIGS.
15-18 and as described in the Descriptions of those figures. FIG.
15 is related to FIG. 3; FIG. 16 is related to FIG. 4; FIG. 17 is
related to FIG. 5; and FIG. 18 is related to FIG. 6.
[0205] Experimental procedures were the same as in Example 1,
except for the following:
[0206] Cells and Reagents
[0207] RAW264.7 (ATCC), Vero, HeLa, 3T3, BHK, MDCK, Huh751, and
HEK293T cells were grown in standard DMEM with 10% FBS, 1%
Penicillin/Streptomycin (GIBCO). CEM cells were cultured in RPMI
media supplemented with 10% fetal calf serum (Hyclone) and 1%
Pen/Strep (Invitrogen). Dr. Glen Barber (University of Miami, Fla.)
provided VSV-GFP. MHV68-Luc was provided by Dr. Ren Sun in MIMG in
UCLA. Luciferase activity was measured using firefly luciferase
substrate kit (Promega). LDH Assay and ATP cell viability (Promega)
were done on cells treated with Ch25h-conditioned media and 25HC
according to manufacturer's instructions. FTI276 and GGTI-298
(Sigma) were used at 5-20 .mu.M, consistent with published doses of
prenylation inhibition (Wilson et al., 1998; Liu et al., 2012;
Miller et al., 2012).
[0208] Expression plasmids were obtained from Genecopoeia, Inc.
Doxycycline inducible expression system was purchased from
Clonetec. SREBP2 expression plasmids were gifts from Dr. Elizabeth
Tarling and Dr. Peter Edwards (UCLA). SREBP1 expression plasmids
were gifts from Dr. Steven Bensinger (UCLA).
[0209] Primary Cells and Cell Lines
[0210] Bone marrow was harvested from 6-8 week C57B/L6 mice
(Jackson Labs) and differentiated in DMEM+10% FBS for 7 days with
10 ng/mL of M-CSF or GM-CSF for macrophage (BMM) or dendritic cells
(BMDC), respectively. On day 6 the media was replaced and on day 7
the cells were stimulated with IFNa or IFNa (PBL Interferon
Source). The cells were treated for 2.5 hours and harvested in
Trizol (Invitrogen). The RNA was isolated by isopropanol
precipitation for microarray analyses. For J2 immortalized
macrophages, bone marrow was infected with J2 retrovirus. A
retrovirus expressing v-raf and c-myc expressing cell line was
established (called GG2EE) and grown in RPMI (10 mM Hepes ph7.8,
10% FBS, 1% Pen/strep). Virus containing supernatant was harvested
and filtered through 0.45 .mu.M filter (Palleroni et al., 1991).
For BCR-ABL transformed B-cells were derived by infecting bone
marrow with BCR-ABL retrovirus as described previously (Scherle et
al., 1990). Stable knockdown in RAW264.7 were generated using
pSiren shRNA knockdown system (Clonetec) according to the published
protocol. Knockdown primer sequences are available by request.
Tail-derived fibroblasts were derived by skinning the tails of mice
and incubating them directly in culture dishes in DMEM 10% FBS.
Cells were scraped and re-plated after 7 days.
[0211] Human peripheral blood mononuclear cells (PBMC) were
obtained from the UCLA Virology Core. These cells were cultured in
RPMI Medium 1640 (Invitrogen) containing 10% FBS, 100 units/ml of
Penicillin+ 100 .mu.g/ml of Streptomycin (Pen/Strep, Invitrogen),
and 20 units/ml of interleukin-2 (Roche). PBMC were costimulated
for 3 days with plate-bound anti-CD3 and soluble anti-CD28
antibodies as previously described (Korin and Zack, 1999).
[0212] Ebola, Nipah, RVFV, RSSEV Plaque Assay
[0213] Plaque Assays were performed on Vero cells (for EBOV, Nipah,
and RVFV) in 12-well plates or BHK-SA cells (for RSSEV) in 6 well
plates. Cells were infected for 1 hr at 37.degree. C. with serial
10-fold dilutions of supernatant aliquots from infected cells. The
cells were then overlaid with growth medium containing 0.6%
methylcellulose (for EBOV, Nipah, and RVFV) or 0.5% tragacanth gum
(for RSSEV). After 3 days (RVFV, Nipah), 4 days (RSSEV) and 10 days
(EBOV), cells were fixed with 10% buffered formalin, stained with
crystal violet and plaques counted. All work involving EBOV, Nipah,
RSSEV and wild-type RVFV, were performed at the Robert E. Shope
BSL-4 laboratory at UTMB.
[0214] HIV IIIB Pseudotyped Virus Production
[0215] HIV-IIIB pseudovirus were made of HIV-IIIB envelope on a
NL4-3 backbone coexpressing luciferase (pNL4-3.Luc.-R-E). Plasmids
were obtained through the NIH AIDS and Research and Reference
Reagent Program. Pseudovirus were generated by cotransfection of
293T cells with envelope deleted LucRE-vector and envelope
expressing vector at a 3:1 .mu.g ratio with Bioline Bio T
transfection reagent. 72 hours post transfection viral supernatant
was collected, clarified by low speed centrifugation and stored at
-80 c. The number of infectious virus particles was determined by
serial dilution assay on Ghost HI-X4 cells, cells that express GFP
controlled by a HIV LTR promoter. Briefly, 4 X 104 Ghost HI-X4
cells were seeded into a 48 well dish. 24 hour later, cells are
infected with 2 fold serially diluted pseudovirions. 48 hours
later, cells were collected and the percentage of positive cells
were determined using FACs.
[0216] Liposome Competition Experiment with 25HC and LJ001
[0217] Recombinant unilamillar liposomes with a composition of
7:3
[0218] phosphatidylcholine:cholesterol (Encapsula Inc.) was added
to HEK293T with or without 25HC (1 uM) for 8 h. Cells were washed
with PBS and infected with VSV-GFP for 1 h and quantified by FACs
at 9 hpi. LJ001 treatment was described previously (Wolf et al.,
2010). Briefly, LJ001 was mixed with liposomes and VSV-GFP for 10
min prior to infection because it intercalates into viral membrane
to inhibit fusion.
References for Example 2
[0219] Butler, S. L., Hansen, M. S. T., and Bushman, F. D. (2001).
A quantitative assay for HIV DNA integration in vivo. Nat Med 7,
631-634. [0220] Cavrois, M., de Noronha, C., and Greene, W. C.
(2002). A sensitive and specific enzyme-based assay detecting HIV-1
virion fusion in primary T lymphocytes. Nat Biotech 20, 1151-1154.
[0221] Korin, Y. D., and Zack, J. A. (1999). Nonproductive Human
Immunodeficiency Virus Type 1 Infection in Nucleoside-Treated G0
Lymphocytes. Journal of Virology 73, 6526-6532. [0222] Liu, X. V.,
Ho, S. S. W., Tan, J. J., Kamran, N., and Gasser, S. (2012). Ras
Activation Induces Expression of Raet1 Family NK Receptor Ligands.
The Journal of Immunology 189, 1826-1834. [0223] Miller, B. T.,
Ueta, C. B., Lau, V., Jacomino, K. G., Wasserman, L. M., and Kim,
B. W. (2012). Statins and Downstream Inhibitors of the
Isoprenylation Pathway Increase Type 2 Iodothyronine Deiodinase
Activity. Endocrinology 153, 4039-4048. [0224] Mortazavi, A.,
Williams, B. A., McCue, K., Schaeffer, L., and Wold, B. (2008).
Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat
Meth 5, 621-628. [0225] Palleroni, A. V., Varesio, L., Wright, R.
B., and Brunda, M. J. (1991). Tumoricidal alveolar macrophage and
tumor infiltrating macrophage cell lines. Int. J. Cancer 49,
296-302. [0226] Scherle, P. A., Dorshkind, K., and Witte, O. N.
(1990). Clonal lymphoid progenitor cell lines expressing the
BCR/ABL oncogene retain full differentiative function. Proceedings
of the National Academy of Sciences 87, 1908-1912. [0227] Takada,
A., Robison, C., Goto, H., Sanchez, A., Murti, K. G., Whitt, M. A.,
and Kawaoka, Y. (1997). A system for functional analysis of Ebola
virus glycoprotein. Proceedings of the National Academy of Sciences
94, 14764-14769. [0228] Wang, L., Feng, Z., Wang, X., Wang, X., and
Zhang, X. (2010). DEGseq: an R package for identifying
differentially expressed genes from RNA-seq data. Bioinformatics
26, 136-138. [0229] Wilson, A. L., Erdman, R. A., Castellano, F.,
and Maltese, W. A. (1998). Prenylation of Rab8 GTPase by type I and
type II geranylgeranyl transferases. Biochem. J. 333, 497-504.
[0230] Wolf, M., Wang, Y., Freiberg, A., Aguilar, H., Holbrook, M.,
and Lee, B. (2009). A catalytically and genetically optimized
beta-lactamase-matrix based assay for sensitive, specific, and
higher throughput analysis of native henipavirus entry
characteristics. Virology Journal 6, 119. [0231] Wolf, M. C.,
Freiberg, A. N., Zhang, T., Akyol-Ataman, Z., Grock, A., Hong, P.
W., Li, J., Watson, N. F., Fang, A. Q., Aguilar, H. C., et al.
(2010). A broad-spectrum antiviral targeting entry of enveloped
viruses. Proceedings of the National Academy of Sciences 107,
3157-3162. [0232] Zhang, L., Jiang, Q., Li, G., Jeffrey, J.,
Kovalev, G. I., and Su, L. (2011). Efficient infection and
impairment of pDC in the bone marrow and peripheral lymphoid organs
during early HIV-1 infection in humanized rag2-/-.gamma.C-/- mice
in vivo. Blood.
[0233] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
changes and modifications of the invention to adapt it to various
usage and conditions and to utilize the present invention to its
fullest extent. The preceding preferred specific embodiments are to
be construed as merely illustrative, and not limiting of the scope
of the invention in any way whatsoever. The entire disclosure of
all applications, patents, and publications cited above, including
U.S. Provisional application 61/643,110, filed May 4, 2012, are
hereby incorporated by reference in their entirety, particularly
with regard to the subject matter for which they are cited.
Sequence CWU 1
1
13119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1tgctacaacg gttcggagc 19222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2agaagcccac gtaagtgatg at 22319DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3aagcgaaact ggcggaaac
19420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4taaccgatgt tgggcatcag 20525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gatagtaccg gaggattgac gacta 25625DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 6gatagtaccg gaggattgac
gacta 25720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7tcaaaccatc cgagccattc 20821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
8tgcaccgcca caaggcagag a 21920DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9tgtgtgcccg tctgttgtgt
201020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10gagtcctgcg tcgagagagc 201120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
11cagtggcgcc cgaacaggga 201224DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12acccactccc tcttagccaa tatt
241318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13gtagggctag gcccaccg 18
* * * * *
References