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Antimicrobial Agents and Chemotherapy, May 2000, p. 1236-1241, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Inhibition of Human Rhinovirus-Induced Cytokine
Production by AG7088, a Human Rhinovirus 3C Protease
Inhibitor
L. S.
Zalman,*
M. A.
Brothers,
P. S.
Dragovich,
R.
Zhou,
T. J.
Prins,
S. T.
Worland, and
A. K.
Patick
Agouron Pharmaceuticals, Inc., San Diego,
California 92121
Received 6 August 1999/Returned for modification 18 October
1999/Accepted 7 February 2000
 |
ABSTRACT |
Symptom severity in patients with human rhinovirus (HRV)-induced
respiratory illness is associated with elevated levels of the
inflammatory cytokines interleukin-6 (IL-6) and IL-8. AG7088 is a
novel, irreversible inhibitor of the HRV 3C protease. In this study,
AG7088 was tested for its antiviral activity and ability to inhibit the
production of IL-6 and IL-8 in a human bronchial epithelial cell line,
BEAS-2B. Infection of BEAS-2B cells with HRV 14 resulted in the
production of both infectious virus and the cytokines IL-6 and IL-8.
Treatment of HRV 14-infected cells with AG7088 resulted in a
statistically significant (P, <0.05) dose-dependent
reduction in the levels of infectious virus as well as IL-6 and IL-8
released into the cell supernatant compared to the results obtained for
compound-free infected cells. AG7088 was also able to inhibit the
replication of HRV 2 and 16 in BEAS-2B cells. In time-of-addition
studies, AG7088 could be added as late as 14 to 26 h after HRV 14 infection of BEAS-2B cells and still result in a statistically
significant (P, <0.05) reduction in the levels of
infectious virus, IL-6, and IL-8 compared to the results obtained for
compound-free infected cells. These findings have implications for the
development of an antirhinovirus agent that may not only block virus
replication but also diminish symptoms.
| |
INTRODUCTION |
Human rhinoviruses (HRV), which
include over 100 different virus serotypes, are responsible for a
significant portion of common colds experienced each year (reviewed in
references 7, 26, and 34). In
patients with underlying respiratory disorders, HRV infections may lead
to sinusitis, otitis media, and lower-respiratory-tract illnesses and
also may lead to exacerbations of asthma, cystic fibrosis, and
bronchitis (3). Many of the clinical symptoms observed in
patients with HRV-induced respiratory illness, such as sore throat,
nasal congestion, sneezing, and runny nose, are associated with
elevated cytokine levels that can be detected in nasal washings
(27, 31, 33). In addition, experimental HRV infections have
been shown to produce an increase in the levels of one or more of the
inflammatory cytokines and mediators, including interleukin-6 (IL-6),
IL-8, IL-1
, kinins, tumor necrosis factor, histamine, and soluble
intercellular adhesion molecule 1 (ICAM-1) (32, 38,
45; R. B. Turner, K. Weingand, C. H. Yeh, et al. Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother. abstr. H-48,
1996). Increased levels of both IL-6 (45) and IL-8
(39; Turner et al., 36th ICAAC) have been associated
with symptom severity in HRV-infected patients.
Although a number of compounds have shown in vitro activity against
HRV, no antiviral agent has been shown to be effective against disease
caused by HRV infection in vivo (1, 2, 8, 15, 17, 28). To
date, only one agent, pirodavir, has been shown to be effective when
administered at the time of HRV challenge but not when given 24 h
after HRV infection (19). Recent reports have described a
new class of compounds directed toward a novel target, the HRV 3C
protease (10-14, 20, 22-25, 29, 35-37, 40-42, 44). The
HRV 3C protease is an enzyme responsible for the posttranslational cleavage of viral precursor polyproteins into their mature forms. AG7088 is a novel, irreversible inhibitor of HRV 3C protease which potently inhibits cytopathic effects induced in H1-HeLa cells by all
HRV serotypes tested (48 of 48), with a mean 50% effective concentration (EC50) of 0.023 µM, a mean EC90
of 0.082 µM, and no cytotoxicity observed up to 1,000 µM
(30). The EC50 refers to the concentration of
compound that increased the percentage of live cells, as measured by
formazan production, to 50% that of uninfected, non-compound-treated
cells. In this study, we have extended these findings and have
evaluated the ability of AG7088 to inhibit virus replication as well as
cytokine production in a human bronchial epithelial cell line, BEAS-2B.
| |
MATERIALS AND METHODS |
Compound.
AG7088 was synthesized at Agouron Pharmaceuticals, Inc.
Cells and virus strains.
All HRV serotypes were purchased
from the American Type Culture Collection (ATCC), Manassas, Va. HRV
stocks were propagated in H1-HeLa cells (ATCC), and antiviral assays
were performed with BEAS-2B cells (ATCC) incubated at 34°C. H1-HeLa
cells were grown in minimal essential medium (Life Technologies,
Gaithersburg, Md.) with 10% fetal bovine serum (HyClone, Logan, Utah),
and BEAS-2B cells were grown in serum-free bronchial epithelial cell
growth medium (Clonetics, San Diego, Calif.).
Antiviral assay.
Confluent BEAS-2B cells were infected at a
multiplicity of infection (MOI) of 50 for HRV 14 or mock infected with
medium only. After 2 h of incubation, cells were washed to remove
virus inoculum and resuspended in medium containing appropriate
concentrations of compound or medium only. The compound was added to
the cells at the time of virus infection. After 3 days of infection,
the cell supernatants were removed, clarified by centrifugation (2 min
at 16,000 × g and 20°C), and either stored at
70°C for subsequent use or analyzed immediately for IL-6 and IL-8
by an enzyme-linked immunosorbent assay (ELISA) and for infectious
virus by a virus yield assay. To determine the amount of intracellular
virus present, the cells were washed two times, frozen and thawed two
times, sonicated, clarified by centrifugation (2 min at
16,000 × g and 20°C), and either stored at 70°C
for subsequent use or analyzed immediately for infectious virus. In
certain experiments, uninfected BEAS-2B cells were treated with 50 µg
of Escherichia coli lipopolysaccharide (LPS) (Sigma, St.
Louis, Mo.) per ml for 2 h, washed two times, and incubated with
compound for 3 days. Cell supernatants were analyzed for the presence
of IL-8.
Cell cytotoxicity assay.
The cell cytotoxicity of AG7088 was
measured by a dye reduction method (43). Briefly, BEAS-2B
cells were resuspended at 5 × 104 cells per ml in
medium containing appropriate concentrations of compound or medium
only. Three days later,
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) (Sigma)-phenazine methosulfate (Sigma) was added to the test
plates, and the amount of formazan produced was quantified spectrophotometrically at 450 and 650 nm. Data were expressed as the
percentage of formazan produced in compound-treated cells compared to
that produced in compound-free cells. The 50% cytotoxic concentration
was calculated as the concentration of compound that decreased the
percentage of formazan produced in compound-treated cells to 50% that
produced in compound-free cells.
Time-of-addition assay.
Confluent monolayers of BEAS-2B
cells were infected with HRV at an MOI of 30 or mock infected with
medium only. After 2 hours of virus adsorption, the monolayers were
washed two times with medium and replenished with fresh medium. AG7088
was added at a concentration (10 µM) that was at least 10-fold above
that needed to completely inhibit HRV 14 replication in BEAS-2B cells,
either before virus infection or at various time points thereafter.
Following 3 days of infection, cell supernatants were removed,
clarified by centrifugation (2 minutes at 16,000 × g
and 20°C), and either stored at 70°C for subsequent use or
analyzed immediately for IL-6 and IL-8 content and for infectious virus.
Virus yield assay.
Infectious virus titers were determined
by a virus plaque assay. Briefly, 0.2 ml of serial 10-fold dilutions of
virus was allowed to adsorb to monolayers of H1-HeLa cells. After
1 h of incubation, the cell monolayers were washed twice with
phosphate-buffered saline and overlaid with medium containing 0.5%
SeaPlaque agarose (FMC Bioproducts, Rockland, Maine). After 3 days of
incubation, the cell monolayers were fixed with EAF (65% ethanol, 22%
acetic acid, 4% formaldehyde) and stained with 1% crystal violet, and virus plaques were enumerated. Data were expressed as PFU per milliliter.
ELISA.
Levels of both IL-6 and IL-8 were determined using a
Quantikine ELISA kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions. Data were expressed in picograms per
milliliter and were derived by extrapolation from a standard curve that
was generated in parallel with each experiment. The concentrations of
each cytokine obtained in compound-treated infected cells were
corrected by subtracting the concentrations of each cytokine obtained
in compound-free uninfected cells.
Statistical analysis.
Statistical significance was
determined with a one-way analysis of variance (SAS version 6.12; SAS
Institute Inc., Cary, N.C.).
| |
RESULTS |
HRV 14 infection of BEAS-2B cells.
BEAS-2B cells were
initially infected with a high MOI of HRV 14 to evaluate the time
course of virus production during a single cycle of virus replication.
Following an eclipse phase of approximately 4 h, levels of
infectious virus released into the cell supernatant increased until
reaching a plateau at 24 h after infection (Fig. 1). These levels of infectious virus were
maintained throughout the 72-h time period studied. Comparable levels
of infectious virus in cellular lysates were also detected after
72 h of infection (data not shown). Microscopic evaluation of
infected BEAS-2B cells revealed a lack of virus-induced cytopathology;
cells remained viable during the entire 72-h time period studied.
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FIG. 1.
HRV 14 production in BEAS-2B cells. BEAS-2B cells were
infected with HRV 14 at an MOI of 30, and levels of infectious virus
were determined at various times after infection (hours) as described
in Materials and Methods. Data represent the mean of duplicate or
triplicate determinations.
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|
Levels of IL-6 and IL-8 released into the cell supernatant were assayed
in parallel. In contrast to the levels of infectious
virus, which
increased until reaching a plateau at 24 h after
infection, the
levels of IL-6 and IL-8 continued to increase throughout
the 72-h time
period studied (Fig.
2). In some
experiments, BEAS-2B
cells were infected with HRV 2 and HRV 16. Levels
of infectious
virus as well as IL-6 and IL-8 comparable to those
observed in
HRV 14-infected cells were detected (data not shown).
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FIG. 2.
Cytokine production in HRV 14-infected BEAS-2B cells.
BEAS-2B cells were infected with HRV 14 at an MOI of 30 or mock
infected with medium only, and levels of the cytokines IL-6 and IL-8
were determined at various times after virus infection (hours) as
described in Materials and Methods. Data represent the mean of
duplicate or triplicate determinations.
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|
IL-6 and IL-8 were also produced in uninfected BEAS-2B cell
supernatants. These levels were significantly lower (two- to fivefold)
at 72 h than those detected in infected cell supernatants. In
addition, in contrast to the levels produced in infected cells,
which
continued to increase throughout the 72-h time period studied,
the
levels in uninfected cells increased slowly until reaching
a plateau at
24
h.
Antiviral activity of AG7088 against HRV infection of BEAS-2B
cells.
The antiviral activity and cytotoxicity of AG7088 were
evaluated by use of BEAS-2B cells infected with HRV 14. The results indicated that AG7088 was able to produce a statistically significant (P, <0.05) dose-dependent reduction in the levels of
infectious virus released into the cell supernatant compared to the
results for compound-free infected cells (Fig.
3). Potent antiviral activity was
observed, with concentrations of AG7088 of at least 0.1 µM being
sufficient to produce a greater than 99.9% reduction in the levels of
infectious virus released into the medium compared to the levels of
infectious virus (5.15 ± 0.69 log10 PFU/ml) produced in compound-free infected cells. AG7088 was also able to produce a
statistically significant reduction in the levels of intracellular virus compared to those in compound-free infected cells (data not
shown). No cytotoxicity was observed with AG7088 up to the tested
concentration of 320 µM in BEAS-2B cells by use of the XTT dye
reduction method (data not shown).
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FIG. 3.
Activity of AG7088 against HRV 14 infection in BEAS-2B
cells. BEAS-2B cells were infected with HRV 14 at an MOI of 50 and
incubated with various concentrations of AG7088. Concentrations of the
cytokines IL-6 ( ) and IL-8 ( ) and levels of infectious virus
(log10 PFU/ml) ( ) were determined as described in
Materials and Methods and are expressed as the mean ± standard
deviation of triplicate determinations. The levels of infectious virus
( ),
IL-6 ( ), and IL-8 ( ) found in compound-free infected cells are
shown. The IL-6 and IL-8 values were corrected for the cytokine levels
found in compound-free uninfected cells as described in Materials and
Methods. Statistical significance (P, <0.05; *) was
determined by comparing each cytokine or infectious virus value to that
obtained in compound-free infected cells.
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|
Concomitant with the decrease in the levels of infectious virus, AG7088
was also able to inhibit the HRV-induced production
of IL-6 and IL-8 in
a dose-dependent manner (Fig.
3). In these
experiments, concentrations
of at least 0.1 µM were sufficient
to cause a statistically
significant (
P, <0.05) decrease in the
levels of both
cytokines compared to those in compound-free infected
cells. This
effect was specific for virus-infected cells, since
AG7088 did not
inhibit IL-8 production induced by LPS treatment
or inhibit IL-8
production in uninfected BEAS-2B cells (data not
shown). AG7088 also
demonstrated comparable activity against the
replication of HRV 2 and
HRV 16 as well as against HRV 2- and
HRV 16-induced production of IL-6
and IL-8 (data not
shown).
Time-of-addition assay.
The ability of AG7088 to inhibit virus
replication and HRV-induced cytokine production when added at various
times during a single cycle of virus replication was also evaluated.
Results indicated that AG7088 could be added as late as 26 h after
virus infection and still achieve a statistically significant (P,
<0.05) reduction in the levels of infectious virus compared to
those in compound-free infected cells (Fig.
4A). Likewise, AG7088 could be added up
to 14 and 26 h after virus infection and produce a statistically
significant (P, <0.05) reduction in the amounts of IL-6 and
IL-8, respectively, compared to those in compound-free infected cells
(Fig. 4B).
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FIG. 4.
Time-of-addition assay showing the effects on infectious
virus replication and cytokine production. BEAS-2B cells were infected
with HRV 14 at an MOI of 30, and AG7088 (10 µM) was added at various
times before and after virus infection as indicated (hours). Following
3 days of infection, levels of infectious virus (A) or IL-6 and IL-8
(B) were determined as described in Materials and Methods. Data
represent the mean ± standard deviation of triplicate
determinations. The levels of IL-6 and IL-8 were corrected for the
levels found in compound-free uninfected cells as described in
Materials and Methods. Levels of infectious virus (A) or IL-6 and IL-8
(B) found in compound-free infected cells are designated ctrl.
Statistical significance (P, <0.05; *) was determined by
comparing each value to that obtained in compound-free infected
cells.
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| |
DISCUSSION |
The symptoms associated with both natural and experimental
rhinovirus infections, such as sneezing, nasal congestion, sore throat,
cough, headache, and malaise, begin shortly after infection and follow
the course of virus replication (9). These symptoms are
associated with a number of inflammatory mediators, such as histamine,
bradykinin, prostaglandins, IL-1, IL-6, IL-8, tumor necrosis factor,
kinins, and soluble ICAM-1 (27, 31, 33). IL-6 and IL-8 have
been shown to be associated with symptom severity in both natural and
experimental rhinovirus infections (39, 45; Turner
et al., 36th ICAAC). In this study, we evaluated the ability of an HRV
3C protease inhibitor, AG7088, to inhibit HRV replication and
HRV-induced cytokine production in a human bronchial epithelial cell
line, BEAS-2B. In vivo, nasal epithelial cells represent the target
host cell for HRV replication (5). Although BEAS-2B cells
are bronchial epithelial cells that have been transformed with an
adenovirus type 12-simian virus 40 hybrid, they share many properties
in common with normal respiratory epithelial cells and thus represent a
biologically relevant system for studying HRV infections in vitro
(38).
In these experiments, infection of BEAS-2B cells with HRV resulted in
the production of significant levels of both infectious virus and the
inflammatory cytokines IL-6 and IL-8, while cell viability was
maintained throughout the time period studied. These findings are
consistent with previous studies that have shown that productive
infection of BEAS-2B cells or A549 cells, a cell line of epithelial
origin, with HRV results in the production of IL-6 and/or IL-8 in the
absence of cell cytopathic effects (6, 21, 38). The
continued viability of BEAS-2B cells is in contrast to the complete
destruction of H1-HeLa cells observed during an HRV infection in vitro
(30). It is not clear which of these two pathways is
followed during an HRV infection in patients. In one study, although a
few small foci of cell destruction in the nasal epithelium were
observed, the majority of the cell layer still appeared intact
(4).
AG7088 not only demonstrated potent antiviral activity in inhibiting
HRV 14 replication in BEAS-2B cells but was also efficacious against
the replication of other HRV serotypes tested. These results are
consistent with data generated in H1-HeLa cells, in which AG7088
demonstrated antiviral activity against all HRV serotypes tested, but
are in contrast to data obtained for capsid binding inhibitors, which
demonstrated extensive variability in antiviral activity against HRV
serotypes (1, 28, 30).
AG7088 was also able to concomitantly reduce the levels of both
inflammatory cytokines, IL-6 and IL-8. The inhibition was specific for
virus infection, since the compound had no effect on the cytokines
induced in LPS-treated BEAS-2B cells or produced by uninfected cells.
This result is consistent with studies demonstrating a reduction of
IL-8 levels after inhibition of HRV infection in BEAS-2B cells by an
antibody to ICAM-1 (38). The relevance of the ability of
AG7088 to inhibit virus replication and cytokine production in vitro
can be ascertained from recent human clinical trials with zamanivir, a
sialic acid analogue with activity against influenza virus infection
(18). In these studies, treatment with zamanivir delivered
intravenously prior to viral challenge caused a statistically
significant reduction in both upper-respiratory-tract symptoms and
cytokine levels (16; D. P. Calfee, A. W. Peng, L. M. R. Cass, M. Lobo, and F. G. Hayden, Abstr.
38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. H58, 1998;
R. S. Fritz, F. G. Hayden, D. P. Clafee, L. M. R. Cass, A. W. Peng, W. G. Alvord, and S. E. Straus,
Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. H57, 1998).
The efficacy of AG7088 when its addition was delayed until several
hours after virus infection was demonstrated in a time-of-addition assay. The results showing that AG7088 was still active even when it
was added late in the infection cycle are consistent with the requirement for 3C protease activity throughout the virus life cycle
(30). The evaluation of AG7088 as an antiviral compound in
human clinical trials has recently begun. The finding that AG7088 is
able to inhibit HRV-induced cytokine production when added throughout
the virus life cycle in vitro indicates that the compound not only may
be effective when administered prophylactically but also may be
effective therapeutically when administered after symptoms have begun.
| |
ACKNOWLEDGMENTS |
We thank Min Zhang for helping with the statistical analysis and
Jules Beardsley for help in preparation of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology, Agouron Pharmaceuticals, Inc., 4245 Sorrento Valley Blvd., San Diego, CA 92121. Phone: (619) 622-3062. Fax: (619) 622-5999. E-mail: zalman{at}agouron.com.
| |
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1236-1241, Vol. 44, No. 5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
