Interferon-Induced Expression of MxA in the
Respiratory Tract of Rhesus Macaques Is
Suppressed by Influenza Virus Replication
This information is current as
of June 18, 2017.
Timothy D. Carroll, Shannon R. Matzinger, Meritxell
Genescà, Linda Fritts, Roxana Colòn, Michael B.
McChesney and Christopher J. Miller
J Immunol 2008; 180:2385-2395; ;
doi: 10.4049/jimmunol.180.4.2385
http://www.jimmunol.org/content/180/4/2385
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References
The Journal of Immunology
Interferon-Induced Expression of MxA in the Respiratory
Tract of Rhesus Macaques Is Suppressed by Influenza Virus
Replication1
Timothy D. Carroll,*† Shannon R. Matzinger,*† Meritxell Genescà,*† Linda Fritts,*†
Roxana Colòn,* Michael B. McChesney,*‡ and Christopher J. Miller2*†§
S
easonal influenza A virus is a highly contagious, acute
respiratory tract infection that causes substantial morbidity
and mortality, particularly among young, old, and immunocompromised individuals (1). Innate immune responses are especially critical for controlling influenza virus replication. IFNstimulated genes (ISGs)3 play a central role in innate antiviral
immunity (2). Among the ISGs, myxovirus resistance (Mx) proteins were originally identified as factors conferring resistance to
lethal influenza A virus infections in mice (3). The human Mx
homologue MxA is able to inhibit the replication of orthomyxoviruses and other RNA viruses (reviewed in Ref. 4). MxA expression and MxA-mediated resistance to the influenza virus is associated with IFN-␣␤-dependent, IFN-␣␤-stimulated response
element (ISRE) induction (5), and MxA expression during influenza virus infection requires type I and type III IFN activity (5).
Despite the clear and critical role of Mx1 in anti-influenza immunity in mice and in human cells in vitro, the role of MxA in controlling influenza in primates, including humans, is unknown.
*California National Primate Research Center, †Center for Comparative Medicine,
‡
Department of Pathology and Laboratory Medicine, School of Medicine, and §Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616
Received for publication September 26, 2007. Accepted for publication November
30, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants P51 RR11069, U01
AI57264, and U01 AI5726.
2
Address correspondence and reprint requests to Dr. Christopher J. Miller, California
National Primate Research Center, University of California, One Shields Avenue,
Davis, CA 95616. E-mail address: [email protected]
3
Abbreviations used in this paper: ISG, IFN-stimulated gene; CPE, cytopathic effect;
HI, hemagglutinin inhibition; IP-10, IFN-inducible protein 10; ISRE, IFN-␣␤-stimulated response element; MDCK, Madin-Darby canine kidney; Mx, myxovirus-resistance protein; OAS, 2⬘-5⬘-oligoadenylate synthetase; PI, postinfection; TCID50, 50%
tissue culture infectious dose; vRNA, viral RNA.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
The neuraminidase protein, which is one of the two glycoproteins expressed on the surface of the influenza virion, promotes
both endocytic entry into target cells during the initial stage of
virus infection and the release of the progeny virus by cleavage of
surface sialic acid on the infected cell and progeny virions (6 – 8).
Prophylaxis or treatment of individuals with the neuraminidase
inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza) can effectively reduce influenza virus replication and shedding and moderate the symptoms associated with the disease in humans (9, 10).
The ability of oseltamivir to reduce viral replication can be used
as a tool to define the relationship between influenza viral replication and antiviral immune responses. Oseltamivir prophylaxis
does not affect the generation of hemagglutinin-inhibiting Ab responses (11) in humans or that of neutralizing Ab responses in
mice, although there were decreased cytotoxic T lymphocyte responses in treated mice (12). The relatively normal adaptive immune response in treated individuals indicates that, despite little
detectable virus replication in nasal cavities, sufficient influenza
virus replication occurs throughout the respiratory system to produce a normal Ab response. However, changes in the innate antiviral immune response due to the oseltamivir-mediated reduction
in viral replication have not been reported.
Macaque monkeys are excellent animal models of the human
immune system and they have been used recently to characterize
the innate and adaptive immune response to influenza A virus infection (13, 14). Thus, to determine the effect of reduced influenza
virus replication on innate antiviral immune responses, we treated
rhesus monkeys with oseltamivir before and after inoculation with
influenza A virus. As expected, we found that prophylactic treatment of rhesus monkeys with oseltamivir reduces influenza virus
replication in the tracheas/airways. In the tracheas of both treated
and untreated monkeys, the mRNA levels of most innate antiviral
molecules in the IFN-␣␤ pathway were dramatically increased by
24 h postinfection (PI). However, we also found that the mRNA
level of a single ISG, MxA, was low in the tracheas of untreated
monkeys despite high levels of virus and IFN-␣ mRNA. In marked
contrast, in the tracheas of treated animals the virus levels were
relatively low, the IFN-␣ mRNA levels were elevated, and there
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
To determine the relationship between influenza A virus replication and innate antiviral immune responses, rhesus monkeys were
given oseltamivir before influenza A/Memphis/7/01 (H1N1) challenge. We found that oseltamivir treatment significantly reduced
viral replication in the trachea (p < 0.029). Further, in the trachea of both treated and untreated monkeys the mRNA levels of
most innate antiviral molecules in the IFN-␣␤ pathway were dramatically increased by 24 h postinfection. However, the mRNA
level of a single IFN-stimulated gene, MxA (myxovirus resistance A), the IFN-stimulated gene known to be critical in blocking
influenza virus replication, was significantly lower in the tracheal lavages of untreated monkeys than in the oseltamivir-treated
monkeys (p ⴝ 0.05). These results demonstrate for the first time that uncontrolled influenza A virus replication actively suppresses
MxA gene expression and emphasize the critical role of innate immunity in controlling influenza virus replication in vivo. The
Journal of Immunology, 2008, 180: 2385–2395.
2386
were significantly higher levels of MxA mRNA than in the tracheas
of untreated monkeys. Thus, in primates, virus replication actively
suppresses MxA expression at a major site of influenza A virus
infection.
Materials and Methods
Animals
All animals used in this study were adult rhesus macaques (Macaca mulatta) that were housed at the California National Primate Research Center
(Davis, CA) in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care International standards. When necessary, animals were anesthetized with ketamine hydrochloride (10 mg/kg body weight; Parke-Davis) or 0.7-mg/kg tiletamine
HCl and zolazepam (Telazo; Fort Dodge Animal Health) injected i.m. Animals with prechallenge hemagglutinin inhibition (HI) titers to A/Memphis/7/2001 greater than 1/8 were excluded from the study.
Virus stock production
Animal inoculation groups and sampling
On day 0, animals were inoculated with either the live or heat-killed virus
stock. The inoculum consisted of a either 1 or 6 ml of virus stock instilled
into the trachea, 1 ml of virus stock dripped intranasally, and a drop of
virus stock in each conjunctiva. On days ⫺4, 1, 2, 3, 7, and 14 PI, tracheal
washes were collected. Under ketamine anesthesia, either an 8- or a
5-French pediatric feeding tube (Kendall) was inserted into the trachea
with the aid of a laryngoscope, and an effort was made to place the tip of
the tube several centimeters cranial to the carina. Twelve milliliters of
sterile PBS were instilled into the trachea, the maximum volume of sample
was aspirated through the feeding tubes, and the samples was transferred to
sample storage tubes on ice. On days ⫺4, 0, 7, 14, and 28 PI, heparinized
blood samples were collected from each animal.
oseltamivir regimen. Animals (not shown) that did not complete the full
oseltamivir treatment schedule were removed from subsequent analysis.
Tracheal lavage sample processing
The tracheal lavage samples were diluted with a solution (51 ␮l/ml sample)
containing 0.3% BSA, 20⫻ antibiotic-antimycotic solution, and 1 mg/ml
gentamicin sulfate. The treated samples were spun at 1,000 ⫻ g for 10 min
and all but 1 ml of the supernatant was removed, stored at ⫺70oC, and
subsequently used to determine infectious virus titer and viral RNA
(vRNA) levels. The 1 ml of supernatant and the cell pellet were immediately processed for RNA isolation.
Before antibiotic treatment, a 250 ␮l aliquot of the fresh tracheal aspirate was removed and diluted 1/1 in PBS. This aliquot was spun at 1,000 ⫻
g for 5 min and resuspended in 500 ␮l of 3.8% BSA in PBS. Subsequently,
50 ␮l of the diluted sample was added to 250 ␮l of the 3.8% BSA solution
and spun onto glass slides at 900 rpm in a Shandon Cytospin 3 (Thermo
Electron) for 5 min. The slides were fixed and stained with Wright-Giemsa
Stain.
Virus isolation and quantitation
MDCK cells were cultured in growth medium consisting of MEM (Invitrogen Life Technologies) supplemented with 10% FCS (Gemini Bio-Products). Virus-infected MDCK cells were cultured in infection medium consisting of MEM supplemented with 0.3% BSA fraction V solution
(Invitrogen Life Technologies), 2 mM L-glutamine (Invitrogen Life Technologies), 0.1 mM MEM nonessential amino acid solution (Invitrogen Life
Technologies), 1⫻ antibiotic antimycotic solution (Sigma-Aldrich), 50
␮g/ml gentamicin sulfate (MP Biomedical, Irvine, CA) and 2 ␮g/ml trypsin from bovine pancreas (Sigma-Aldrich). A solution containing 0.3%
BSA, 20⫻ antibiotic-antimycotic solution (Sigma-Aldrich), and 1 mg/ml
gentamicin sulfate was added to tracheal samples before culture.
The endpoint dilution culture assays to determine the TCID50 dose in
previously frozen samples were conducted in 24-flat well tissue culture
plates seeded with MDCK cells at a concentration of 1 ⫻ 105 cells/well.
Cells were allowed to expand in growth medium for 3 days before thawed
samples were added to the wells. On the day of sample inoculation, the
plates were washed twice with PBS and 100 ␮l of a given sample dilution
was added to each well. After 1 h, 900 ␮l of infecting medium was added
to each well. At day 3 PI, each well was observed for the presence of
cytopathic effects (CPEs).
Influenza virus RNA PCR
Two approaches were used to determine body temperature in animals in
different experiments and temperatures were collected from all animals at
approximately the same time of day to account for changes in temperature
due to circadian rhythm. In the experiment to determine the effect of virus
inoculum size on viral replication, body temperature was determined using
IPTT-300 transponders (Bio Medic Data Systems) inserted s.c. in the interscapular region. From day ⫺3 through day 5 PI, temperatures were
recorded twice daily in the morning and afternoon (⬃9 a.m. and 3 p.m.).
The a.m. and p.m. temperatures are expressed as the change in degrees
(⌬°F) in an individual monkey’s temperature as it relates to either the day
⫺3 PI a.m. or p.m. value; changes in the a.m. and p.m. temperature values
are calculated separately. In the experiments to determine the effect of viral
replication on the host immune response, rectal temperatures were collected from sedated animals on the morning of days ⫺4, 1, 2, 3, 7, and 14
PI. Temperatures are expressed as the ⌬°F in an individual monkey’s temperature as it relates to the day ⫺4 PI value.
Before RNA isolation, fresh tracheal lavage was centrifuged at 1,000 ⫻ g
for 10 min. Then 1 ml of supernatant and the cellular pellet were processed
as described below. To determine the level of virion-associated RNA in
samples, the supernatant was removed and lysed at a 5:1 TRIzol LS (Invitrogen Life Technologies) to supernatant ratio. Total RNA was isolated
with TRIzol LS according to the manufacturer’s instructions for RNA
stored in biological fluids. cDNA was prepared using random hexamer
primers (Amersham Biosciences) and SuperScript III reverse transcriptase
(Invitrogen Life Technologies).
A real-time RT-PCR assay on a Prism 7900 sequence detector system
(Applied Biosystems) was used for quantitation of influenza RNA in secretions. A previously described influenza A virus matrix gene-specific
PCR primer set was used for PCR amplification (16). The forward primer
was 5⬘-AGATGAGTCTTCTAACCGAGGTCG-3⬘, the reverse primer was
5⬘-TGCAAAAACATCTTCAAGTCTCTG-3⬘, and the probe was 5⬘TCAGGCCCCCTCAAAGCCGA-3⬘, producing a 100-bp amplicon. The
nominal influenza A matrix copy numbers were determined by a modification of a method previously described (17). Briefly, the copy number of
matrix gene was determined by interpolation of the average measured
threshold cycle number onto a standard curve produced with a purified
plasmid containing a fragment of the M1 gene cloned from the A/Memphis/7/2001 stock. Quantification of the purified plasmid was based on A260
measurements.
Oseltamivir treatment
Tracheal sample cytokine mRNA expression levels
The recommended oral dose of oseltamivir phosphate oral suspension for
pediatric patients having ⬍15 kg of body weight is 30 mg twice daily for
five consecutive days (Tamiflu product information; Roche Pharmaceuticals). Animals selected for prophylactic oseltamivir orally received 35 mg
twice daily on days ⫺1, 0, 1, 2, and 3 PI. On day ⫺1 and the afternoon of
days 0, 1, 2, and 3 PI, the treatments were delivered in fruit treats. On the
mornings of days 0, 1, 2, and 3 PI, oseltamivir was suspended in 2 ml of
PBS and delivered by oral-gastric intubation while the animal was sedated.
Observation logs were kept to determine whether animals completed the
For assessment of host gene expression, total RNA was isolated from the
cellular pellets with TRIzol (Invitrogen Life Technologies) according to
the manufacturer’s instructions. RNA samples were DNase-treated with
DNA-free (Ambion) for 1 h at 37°C. cDNA was prepared using random
hexamer primers (Amersham Biosciences) and SuperScript III reverse
transcriptase (Invitrogen Life Technologies).
Cytokine mRNA levels were determined by RT-PCR as described previously (18, 19). Briefly, the GAPDH housekeeping gene and the target
gene from each sample were run in parallel in the same plate. The reaction
Assessment of body temperature
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The human influenza A virus isolate used in this study, A/Memphis/7/2001
(H1N1), was generously provided by R. Webby at St. Jude Children’s
Research Hospital, Memphis, TN. This isolate was isolated on MadinDarby canine kidney (MDCK) and was not passaged further before expansion in MDCK cells (American Type Culture Collection) to produce the
virus stock used for animal inoculations. The virus stock had a titer of 106.5
TCID50/ml (where TCID50 is 50% tissue culture infectious dose) on
MDCK cells using the method of Reed and Muench (15). To produce the
heat-killed virus used for inoculations, aliquots of the stock were heated in
a 56oC waterbath for 1 h, frozen at ⫺80°C overnight, and then heated again
at 56oC for 1 h just before animal inoculation. This procedure resulted in
complete inactivation of the virus, as we were unable to isolate an infectious virus from treated aliquots of the stock (data not shown).
INFLUENZA A SUPPRESSES MxA
2387
)
The Journal of Immunology
7
7
A
6
5
5
4
4
3
3
2
2
1
1
0
0
B
6
2
4
6
8
10
12
14
16
0
0
2
4
Days PI
10 7
4096
C
10 6
8 10
Days PI
12
14
16
D
2048
1024
512
10
5
10
4
256
128
64
32
16
10 3
8
4
10 2
0
7
14
21
28
35
Days PI
6
5
4
2
0
7
14
21
Days PI
28
35
E
3
2
1
0
-1
-2
-3
-72 -48 -24
0
24 48 72 96 120
Hours PI
was conducted in a 96-well optical plate (Applied Biosystems) in a 25 ␮l
reaction volume containing 5 ␮l of cDNA plus 20 ␮l of Master Mix (Applied Biosystems). All sequences were amplified using the 7900 default
amplification program: 2 min at 50oC, 10 min at 95oC, followed by 40 to
45 cycles of 15 s at 95oC and 1 min at 60oC. The results were analyzed with
the SDS 7900 system software, version 2.1 (Applied Biosystems). Cytokine mRNA expression levels were calculated from normalized ⌬CT
(threshold cycle) values. CT values correspond to the cycle number at
which the fluorescence, due to enrichment of the PCR product, reaches
significant levels above the background fluorescence (threshold). In this
analysis, the CT value for the housekeeping gene (GAPDH) is subtracted
from the CT value of the target (cytokine) gene (⌬CT). In general, the ⌬CT
value for the influenza A-infected sample is then subtracted from the preinfection ⌬CT value (⌬⌬CT). Assuming that the target gene (cytokine) and
the reference gene (GAPDH) are amplified with the same efficiency (data
not shown), the increase in cytokine mRNA levels in test samples is then
calculated as follows: increase ⫽ 2 ⫺⌬⌬CT (user bulletin no. 2 for the ABI
Prism 7700 sequence detection system from Applied Biosystems). Cytokine mRNA levels are expressed as the increase relative to the level for that
cytokine in the individual monkey’s pretreatment lavage sample. Because
the mRNA expression level of housekeeping genes such as GAPDH can
change under activating conditions, we were careful to use the same input
amount of RNA for untreated and experimental samples in the PCR. Samples with the same input amount of RNA consistently resulted in similar
PCR amplification (CT) values for GAPDH. Therefore, it seems that
GAPDH was not significantly up-regulated in response to the treatments
used in this study.
Influenza Ab ELISA
All plasma samples were initially tested for anti-influenza Abs in a 1/800
dilution-screening assay. Results of the screening assay were calculated
using the following ratio: change in OD (⌬OD)/cutoff, where ⌬OD is defined as the difference between the mean OD of a diluted sample tested in
two influenza Ag-coated wells and the mean OD of the same diluted sample tested in two egg Ag-coated wells. The cutoff value is the mean ⌬OD
of two preinfection time points of a sample plus 3 SD values. If the ⌬OD/
cutoff ratio for a sample was ⬎1.0, the sample was considered to be positive and the titer of anti-influenza Abs was determined.
To determine anti-influenza A Ab titers in plasma samples that were
positive in the screening assay, 96-well plates (Nunc-Immuno Maxisorp
plate II) were coated with either detergent-disrupted A/New Caledonia/
20/99 influenza A (Biodesign International) or whole egg protein (Charles
River Laboratory) at 5 ␮g/ml in 0.1 M Na2CO3/NaHCO3 buffer (pH 9.6)
and blocked with 4% nonfat powdered milk. Samples were serially diluted
(1/2) in duplicate and the plates were incubated overnight at 4oC. The
initial dilution of serum tested was 1/4,000. Ab binding was detected using
100 ␮l of a 1/2,000 dilution of peroxidase-conjugated goat anti-monkey
IgG(Fc) (Accurate Chemicals) for 1 h at 37oC. Plates were developed with
o-phenylenediamine dihydrochloride (Sigma-Aldrich) for 5 min and
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. The effect of a variable A/Memphis/
7/2001 (H1N1) dose on tracheal viral replication and
host response in rhesus monkeys. A, Average infectious viral titers (TCID50) in tracheal lavage. B, Average vRNA copy number (log10 copies/ml) in tracheal lavage. C, Average anti (␣)-whole virus
plasma IgG Ab titer. D, Average anti (␣)-A/Memphis/7/2001 plasma HI Ab titer. E, Average change
in body temperature, where the a.m. and p.m. temperatures are relative to ⫺72 and ⫺60 h PI, respectively. Temperatures were collected every 12 h with
BMDS IPTT-300 transponders. A/Memphis/7/2001
(106.5 TCID50/ml) dosing: f, 6 ml intratracheal/1 ml
intranasal (n ⫽ 3); E, 1 ml intratracheal/1 ml intranasal (n ⫽ 3).
6
2388
stopped with H2SO4 before reading the OD at 490 nm with a background
subtraction of 540 nm. For each serum sample, the endpoint titer of antiinfluenza A Abs was defined as the last dilution giving a ⌬OD value ⬎0.1,
where ⌬OD was defined as the difference between med OD values in
two-influenza Ag-coated and two-egg Ag coated wells.
HI assay
Titers of anti-H1 Abs were calculated using the HI test of hemagglutinin
inhibition. The HI test, originally described by Hirst (20) and later modified
by Salk (21), was performed using the microtiter plate protocol revised by
the World Health Organization (22). Briefly, monkey plasma samples were
treated with receptor-destroying enzyme (RDE) II (VWR International)
according to the manufacturer’s instructions. Treated plasma were then
serially diluted 1/2 in PBS. Twenty-five microliters of standardized whole
virus solution containing four hemagglutinin units/25 ␮l were added to
each well. After a 10-min incubation at room temperature, 50 ␮l of 0.5%
chicken RBC (Colorado Serum) were added to each well. Results were
read after a 1-h incubation. The viral Ag used was the A/Memphis/7/2001
stock virus grown in 10-day-old embryonated chicken eggs.
For intracellular staining to detect influenza-specific T cells in PBMCs, a
modification of a previously reported protocol was used (23). Briefly, frozen samples were thawed and rested overnight at 37°C in 5% CO2 atmosphere in complete RPMI 1640 medium containing 10% FCS. The next
day, cells were adjusted to 1 ⫻ 106/200 ␮l, and incubated with anti-CD28
and anti-CD49d Abs (1 ␮g/ml final concentration; BD Biosciences) as
costimulatory molecules in a total volume of 200 ␮l of complete RPMI
1640 and 10% FCS. In all experiments the following samples were prepared: sucrose-purified (A/New Caledonia/20/1999) H1N1 influenza virus
at 5 ␮g/ml, background controls containing costimulatory molecules, and
a positive control stimulated with staphylococcal enterotoxin B (0.2 ␮g/ml,
Sigma-Aldrich). A mixture of anti-CD107a and anti-CD107b-FITC Abs
(clones H4A3 and H4B4, respectively; BD Pharmingen) was added at a
pretitrated volume. Cells were incubated for 16 h at 37°C and brefeldin A
(Sigma-Aldrich) and monensin (GolgiStop; BD Biosciences) were added
for the last 10 h. Following incubation, cells were washed and surfacestained with anti-CD3-Pacific Blue, anti-CD4-PerCP-Cy5.5, or anti-CD8allophycocyanin-Cy7. A marker to exclude dead cells (7-aminoactinomycin D; Molecular Probes) was added and then samples were fixed (1%
paraformaldehyde), permeabilized (0.5% saponin), and stained intracellularly with IFN-␥-allophycocyanin (clone B27), TNF-␣-PE-Cy7 (clone
Mab11), or IL-2-PE (clone MQ1-17H12) for 20 min at room temperature.
All mAbs were from BD Pharmingen unless specified. After washing
with permeabilizing buffer, cells were fixed in PBS containing 1%
paraformaldehyde.
Data were acquired using a FACSAria flow cytometer (BD Biosciences)
and analyzed using FlowJo software (Tree Star) and a Macintosh G5 computer (Apple). At least 100,000 events in the forward/side scatter lymphocyte gate were acquired. The background level of cytokine staining varied
from sample to sample but was typically ⬍0.05% of the unstimulated
CD8⫹ T lymphocytes. The only samples considered positive were those in
which, after subtracting the background control, there were at least five
positive events for a single functional marker, three positive events for two
or more simultaneous functional markers, and the sum of the different
combinations of responses represented at least 10 events. In addition, a
sample was not considered positive for a particular combination of functions if the frequency of responding T cells responding with that particular
combination of functions was lower than 0.02%. All data are reported after
subtraction of the medium control cultures.
Statistical analysis
To compare the mean TCID50 in tracheal washes of the treated vs untreated
animal groups, a one-way Mann-Whitney U test was used. A one-way
ANOVA test and Bonferroni’s multiple comparison post hoc test were
applied to compare the mean levels of vRNA and host gene mRNA in
tracheal washes in the treated, untreated, and heat-inactivated virus and
naive animal groups. GraphPad Prism version 4.a for Apple OSX10.4
(GraphPad Software) and Macintosh G5 computers (Apple) were used for
all analyses.
A
7
B
8
6
7
6
6
5
4
4
5
2
4
0
3
2
2
1
1
0
0
2
4
6
0
8 10 12 14 16
p < 0.001
p < 0.05
2
4
3
p < 0.001
8
p = 0.029
6
0
0
2
4
6
8 10 12 14 16
DAYS PI
FIGURE 2. The effect of oseltamivir treatment on A/Memphis/7/2001
(H1N1) replication in the trachea. A, Average infectious viral titers
(TCID50) in tracheal lavages. Inset, results of the one-way Mann-Whitney
U test between mean peak tracheal lavage TCID50 of untreated and oseltamivir-treated rhesus. B, Average vRNA copy number (log10 copies/ml) in
tracheal lavages. Inset, Graph of one-way ANOVA with Bonferroni’s multiple comparison post hoc test comparing the average peak of tracheal
lavage vRNA of untreated, oseltamivir-treated, and heat-inactivated influenza virus animals. f, Untreated (n ⫽ 3); E, oseltamivir-treated (n ⫽ 4);
and Œ, heat-inactivated virus (n ⫽ 4).
Results
The effect of influenza virus inoculum size on virus replication,
body temperature, and anti-influenza Ab responses
To determine the effect of inoculum size (viral dose) on virus replication in rhesus monkeys, three monkeys were inoculated intratracheally with 6 ml and intranasally with 1 ml of the A/Memphis/
7/2001 (H1N1) stock; this is designated as the high-dose group. A
second group of three monkeys was inoculated intratracheally with
1 ml and intranasally with 1 ml of the same virus stock; this is
designated as the low-dose group. Tracheal lavages were obtained
Table I. Effect of prophylactic oseltamivir on influenza virus
replication in trachea
Peak Viral Levels (Log10)
Animal Groups
TCID50
Peak
Day
vRNA
Peak
Day
Untreated
33073
33178
34421
Mean ⫾ SEM
4.67
3.50
4.50
4.22 ⫾ 0.62a
1
1
1
5.81
5.44
6.40
5.88 ⫾ 0.48b
1
1
2
Oseltamivir treated
31657
31981
32410
33182
Mean ⫾ SEM
1.50
1.50
2.33
2.33
1.92 ⫾ 0.048a
2
1
1
1
4.75
5.05
4.62
5.20
4.96 ⫾ 0.27a,b
2
2
2
1
2.87
3.51
2.43
3.08
2.97 ⫾ 0.45a,b
1
1
1
1
Heat-inactivated virus
31392
31625
31705
31864
Mean ⫾ SEM
0
0
0
0
a
Mean values are significantly different between treated and untreated monkeys
( p ⫽ 0.029; two-tailed unpaired t test).
b
Mean values are significantly different between monkeys that were treated,
untreated, and inoculated with heat-inactivated virus (p ⬍ 0.001; one-way
ANOVA test).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Intracellular cytokine staining for assessing anti-influenza
T cell responses
INFLUENZA A SUPPRESSES MxA
The Journal of Immunology
2389
106
A
105
104
103
102
0
7
14
21
28
35
Days PI
B
0
7
14
21
28
35
Days PI
C
4
3
FIGURE 3. Tracheal lavage cytospins during A/Memphis/7/2001 (H1N1)
infection of an untreated and an oseltamivir-treated rhesus monkey. A, C, E,
and G are from an untreated monkey. B, D, F, and H are from an oseltamivirtreated monkey. Samples from days ⫺4, 1, 2 and 3 are shown for each animal.
Note that healthy ciliated epithelial cells are much more common in the oseltamivir-treated monkey at all time points. Intracytoplasmic inclusion bodies
in influenza A virus-infected epithelial cells and inflammatory cells (neutrophil
and macrophage) are common in the untreated monkey after virus inoculation.
Wright-Giemsa stain was used. Bar equals 20 ␮m.
2
1
0
-1
-2
0
2
4
6
8
Days PI
before inoculation and on days 1, 2, 3, 7, and 14 PI. The levels of
virion-associated influenza virus RNA detected by PCR in the tracheal lavages of the high-dose and low-dose monkey groups were
indistinguishable. Tracheal lavage vRNA levels (⬎106 copies/ml)
peaked at 24 h PI followed by a rapid 3 log10 decline by day 7 PI.
Very little vRNA was detected in tracheal lavages of any animals
on day 14 PI (Fig. 1).
Similarly, the titers of infectious virus isolated from tracheal
samples of the high-dose and low-dose groups were very similar
(Fig. 1). In addition, at 12 h PI both groups had similar spikes in
body temperature that resolved by 96 h PI (Fig. 1). Finally HI titers
and anti-whole virus IgG Ab titers in the high- and low-dose
groups were indistinguishable. All animals had preinoculation HI
titers of 1:4 that dramatically increased by day 14 PI. The IgG Ab
responses to whole influenza virus were identical, with strong responses in all animals by day 14 PI (Fig. 1).
Oseltamivir effectively blocks influenza virus replication in
rhesus monkeys
To determine the effect of prophylactic oseltamivir on influenza
virus replication in rhesus monkeys, four monkeys were treated
with oseltamivir twice daily for 5 days beginning 24 h before in-
FIGURE 4. Body temperature and Ab response during A/Memphis/7/
2001 (H1N1) infection in untreated-, oseltamivir-, and inactivated virusinoculated rhesus monkeys. A, Average whole anti (␣)-A/New Caledonia/
20/1999 (H1N1) plasma IgG Ab titer. B, Average anti (␣)-A/Memphis/7/
2001 plasma HI Ab titer. C, Average change in body temperature (°F)
compared with day 0. Temperatures were collected every 24 h by rectal
thermometer. f, Untreated (n ⫽ 3); E, oseltamivir-treated (n ⫽ 4); and Œ,
heat-inactivated virus (n ⫽ 4) monkeys.
fluenza virus inoculation. The monkeys were inoculated intratracheally with 6 ml and intranasally with 1 ml of the A/Memphis/
7/2001 (H1N1) stock; the concentration of virus was 106.5 TCID50/
ml. The levels of infectious virus and vRNA in tracheal lavages of
the four treated and three untreated monkeys were compared.
In the untreated and oseltamivir-treated animals, influenza RNA
was detectable in the tracheal lavage samples on days 1, 2, and 3
PI (Fig. 2). By day 7 PI, vRNA was still detectable in the samples
from oseltamivir-treated and untreated animals. However, the levels of vRNA in the trachea were different among the groups. Thus,
the mean peak vRNA level in the oseltamivir-treated monkeys was
significantly lower ( p ⬍ 0.05) than the mean peak vRNA level in
the untreated monkeys (Fig. 2 and Table I).
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4096
2048
1024
512
256
128
64
32
16
8
4
2390
INFLUENZA A SUPPRESSES MxA
Table II. Effect of oseltamivir on antibody responses to influenza A
virus infection
Plasma Anti-Influenza Ab Titers
(Reciprocal Mean Log2 Titer)
HI Titers
ELISA Titers
Posta
Untreated
33073
33178
34421
Mean
8
8
8
8
2,048
512
1,024
1,195
256
64
128
149
⬍800
⬍800
⬍800
⬍800
400,000
64,000
80,000
181,333
Oseltamivir treated
31657
31981
32410
33182
Mean
4
8
8
8
7
1,024
1,024
512
1,024
896
256
128
64
128
144
⬍800
⬍800
⬍800
⬍800
⬍800
200,000
100,000
100,000
160,000
140,000
Heat inactivated
31392
31625
31705
31864
Mean
4
4
8
4
5
16
64
32
4
29
4
16
4
1
6
⬍800
⬍800
⬍800
⬍800
⬍800
⬍800
6,400
⬍800
⬍800
1,600
Animal/Group
Prea
Posta
a
Pre represents a time point prior to the day of challenge and Post represents day
28 postchallenge.
The mean titer of infectious virus isolated from tracheal samples
of untreated animals was 104 TCID50/ml, significantly higher than
the mean virus titer in the oseltamivir-treated animals ( p ⫽ 0.029;
Fig. 2 and Table I). Taken together, these data demonstrate that
oseltamivir is effective at blocking viral replication in the lower
respiratory tract of the rhesus monkey.
To determine how much of the vRNA detected in tracheal lavages represented the input inoculum rather than de novo virus
replication, four additional animals were inoculated with the same
dose of heat-killed A/Memphis/7/2001. As expected, the mean
peak vRNA level of the killed virus group was dramatically
(⬃1,000-fold) and significantly lower than those of both the oseltamivir-treated and untreated animal groups (Fig. 2 and Table I).
Infectious virus was not isolated from either tracheal samples collected from the heat-killed virus-inoculated animals (Fig. 2 and
Table I). Thus, in untreated monkeys the input viral inoculum contributed ⬍0.1% to the peak vRNA levels after inoculation of live
virus.
Oseltamivir treatment reduces inflammatory cell infiltrates
associated with influenza infection
Before influenza challenge, tracheal wash cytospins from the oseltamivir-treated and untreated monkeys contained ciliated epithelial and rare inflammatory cells (Fig. 3). At 24 and 48 h PI, the
number of epithelial cells was increased in the tracheal washes of
both treated and untreated monkeys and the CPEs (cellular swelling, cytoplasmic vacuolization, and inclusion bodies) in epithelial
cells induced by influenza virus infection were common in samples
from untreated monkeys (Fig. 3). By 72 h PI, regenerating epithelial cells were common, viral CPEs were rare, and lymphocytes
and neutrophils were common (Fig. 3). CPEs in epithelial cells and
all inflammatory cells were much less numerous in the tracheal
wash samples of the oseltamivir-treated monkeys (Fig. 3).
The monkeys inoculated with heat-killed virus had no change in
body temperature after inoculation but both the untreated and oseltamivir-treated monkeys had a spike in body temperature at 24 h
PI that resolved by 96 h PI (Fig. 4) Although the difference was not
statistically significant, the increase in the peak body temperature
was more marked in the untreated monkeys than in the oseltamivir-treated monkeys. Despite the decreased viral replication in oseltamivir-treated monkeys, HI titers and anti-whole virus IgG Ab
titers were not affected by oseltamivir treatment (Fig. 4 and Table
II). The monkeys inoculated intratracheally and intranasally with
heat-killed virus made HI and anti-whole virus IgG Ab responses
in plasma, although they were relatively weak and delayed compared with the animals inoculated with live virus (Fig. 4).
Anti-influenza T cell responses in untreated and
oseltamivir-treated influenza infection
To assess influenza virus-specific T cell responses, PBMCs were
restimulated with inactivated influenza virions for 16 h and then
permeabilized and stained to detect cytokine production and degranulation. On the day of the experimental inoculation, influenzaspecific CD4⫹ T cell responses were found in seven of 13 animals
and four of 13 animals had influenza-specific CD8⫹ T cell responses on day 0 (Figs. 5 and 6). These responses were easily
detected as the mean frequency of 855 (⫾371 SEM) influenzaspecific CD4⫹ T cells per 105 CD3⫹ T cells and 603 (⫾344 SEM)
influenza-specific CD8⫹ T cells per 105 CD3⫹ T cells. These
day-0 T cell responses were characterized by a large fraction of
TNF-␣-secreting T cells in both subsets, with more IL-2 and less
IFN-␥ secretion in CD4⫹ T cells compared with CD8⫹ T cells
(Figs. 5 and 6A). None of the T cells degranulated upon stimulation. Note that although all four of the oseltamivir-treated animals
were negative for influenza-specific T cell responses on day 0 (Fig.
5), differences in cell viability or the oseltamivir treatment do not
explain the differences in responses, as the day 0 T cells from all
animals made similar responses to staphylococcal enterotoxin B
stimulation (data not shown). Furthermore, three of the four oseltamivir-treated animals with no influenza-specific T cells had HI
titers against the challenge virus of 1:8 (Table II), suggesting
that these animals had prior exposure. The influenza-specific
memory T cells and Ab responses found at day 0 were presumably
induced by prior exposure to an antigenically similar influenza A
virus circulating in the humans and introduced by the animal care
staff. The strength of the T cell responses, relative to the HI titers,
suggests that they are directed against relatively conserved influenza Ags such as matrix and nucleoprotein (NP).
To assess the effect of viral replication on T cell responses to experimental influenza virus inoculation, the influenza-specific T cell
responses of the four oseltamivir-treated animals and the three
matched untreated animals (Table I) on days 15 and 30 PI were compared (Fig. 6B). All seven animals had influenza-specific CD4⫹ T cell
responses on day 15 PI, but one animal in each group did not have
detectable influenza-specific CD8⫹ T cells. By day 30 PI all seven
animals had easily detected antiviral CD4⫹ T and CD8⫹ T cells in
blood. The mean frequency of influenza-specific CD4⫹ and CD8⫹ T
cells was lower in the oseltamivir-treated animals (Fig.6B) at both
time points. It is important to note that on day 0 all three of the control
animals, but none of the oseltamivir-treated animals, had pre-existing
T cell responses. However, efficient expansion of a memory influenza-specific T cell pool in the untreated animals after challenge does
not explain the results, as the frequencies of influenza-specific T cells
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Prea
Fold
Increase
Oseltamivir blunts the body temperature spike associated with
acute influenza infection but has no effect on the generation of
anti-influenza Ab responses
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. Antiviral T cell responses in PBMCs before and after A/Memphis/7/2001 (H1N1) influenza virus infection in untreated and oseltamivir-treated rhesus monkeys. The response to purified inactivated H1N1
influenza virus stimulation is shown. The pie charts in the top panels summarize the CD4⫹ T cell responses on days 0, 15, and 30 PI and the extent to which the CD4⫹ T cell response was polyfunctional. The pie
charts in the bottom panels summarize the T cell responses on days 0, 15 and 30 PI and the extent to which the CD8⫹ T cell response was polyfunctional. Empty circles indicate that there was no positive response
in the indicated T cell subset in that sample. Each portion of a pie chart indicates the percentage of influenza-specific T cells that responded with one, two, three, or four functions, and the arcs around the pie show
the function or combination of functions to which the specific response corresponds (see color legend). “ID sample” indicates the animal number.
The Journal of Immunology
2391
2392
A
INFLUENZA A SUPPRESSES MxA
CD8+ T cells
CD4+ T cells
10
1.48† ± 0.78
A
10 4
3
10 1
10 2
0 days PI
2.06 ± 0.77
10 0
10 1
10 0
B
54*% (7/13)
31% (4/13)
CD4+ T cells
CD8+ T cells
oseltamivir
0.21 ± 0.11
controls
1.17 ± 0.25
100% (4/4)
100% (3/3)
0.58 ± 0.35
1.86 ± 0.28
oseltamivir
15 days PI
30 days PI
B
10 2
0.17 ± 0
controls
1.11 ± 0.36
75% (3/4)
67% (2/3)
0.31 ± 0.16
1.31 ± 0.34
10 -1
10 -1
10 -2
0
10 3
2
4
6
8 10 12 14 16
10 -2
10 2
10 1
10 1
10 0
10 0
10 -1
10 -1
0
2
2
10 3
C
10 2
10 -2
0
4
6
8
10 12 14 16
10 -2
4
6
8
10 12 14 16
6
8
10 12 14 16
6
8 10 12 14 16
D
0
2
4
100% (4/4)
0.58 ± 0.35
100% (3/3)
100% (4/4)
100% (3/3)
10 4
0.82 ± 0.28
CD107ab
IFN-γ
10
3
IL-2
10 2
4
TNF-α
10 1
1
2
FIGURE 6. Mean antiviral T cell responses in PBMCs before and after
A/Memphis/7/2001 (H1N1) influenza virus infection in untreated and oseltamivir-treated rhesus monkeys. The response to purified-inactivated
(H1N1) influenza virus stimulation is shown. A, The pie charts summarize
the mean T cell responses of all 13 animals inoculated with live virus on
day 0 and the extent to which the CD4⫹ and CD8⫹ T cell responses were
polyfunctional. For each pie chart the percentage (and proportion) of responding animals is indicated below the chart. The mean frequency
(⫾SEM) of each response is shown as a white number in the middle of the
pie chart; only animals with a positive response are included. B, The pie
charts summarize the T cell responses in untreated and oseltamivir-treated
rhesus monkeys on days 15 and 30 PI and the extent to which the CD8⫹
T cell response was polyfunctional. For each pie chart the percentage (and
proportion) of responding animals is indicated below the chart. The mean
frequency (⫾SEM) of each response is shown as a white number in the
middle of the pie chart; only animals with a positive response are included.
Each portion of a pie chart indicates the percentage of influenza-specific T
cells that responded with one, two, three, or four functions, and the arcs
around the pie show the function or combination of functions to which the
specific response corresponds (see color legend).
in these animals declined after challenge. The quality of the influenzaspecific CD4⫹ and CD8⫹ T cell responses was variable among animals within groups (Fig. 5).
The decreased viral replication mediated by oseltamivir does
not affect IFN-␣, 2⬘-5⬘-oligoadenylate synthetase (OAS),
IFN- ␥, or IFN-inducible protein 10 (IP-10) mRNA expression,
but MxA mRNA expression is increased
To determine the effect of the oseltamivir-mediated reduction in
viral replication on innate antiviral immune responses, the levels of
mRNA for anti-viral molecules; IFN-␣, IFN-␥, OAS, IP-10, and
MxA in tracheal secretions of the treated animals and untreated
animals were compared. To control for the possibility that oseltamivir directly affects gene expression, the levels of the same innate
immune molecules in the tracheal lavages of five animals treated
with same dose and schedule of oseltamivir but never inoculated
with influenza virus were also determined. Finally, to control for
the possibility that the input vRNA in the inoculum and not viral
3
10 0
E
ANOVA p = 0.0008
F
10 2
p< 0.001
p < 0.05 p < 0.01
10 1
10 0
10 -1
10 -1
10 -2
10 -2
0
2
4
Days PI
FIGURE 7. Innate antiviral responses to A/Memphis/7/2001 (H1N1)
influenza virus infection in untreated, oseltamivir-, and inactivated virusinoculated rhesus monkeys. Responses are reported as the increase in genespecific mRNA levels relative to pre-exposure values from the same animal’s matched pre-exposure sample. A, IFN␣. B, OAS. C, MxA. D, IP-10.
E, A one-way ANOVA with Bonferroni’s multiple comparison post hoc
test was used to determine the significance between the average MxA
mRNA levels in untreated, oseltamivir-treated, heat-inactivated virus, and
oseltamivir-only monkeys. F, IFN-␥. f, Untreated (n ⫽ 3); F, oseltamivirtreated (n ⫽ 4); ‚, heat-inactivated virus (n ⫽ 4); and ⴱ, oseltamivir-only
(no virus; n ⫽ 5) monkeys.
replication was responsible for inducing gene expression, the levels of the same innate immune molecules in the tracheal lavages of
the four monkeys inoculated with the heat-inactivated influenza
were determined.
At 24 h PI, there was a 103- to 104-fold-increase in IFN-␣
mRNA levels in tracheal washes of oseltamivir-treated and untreated monkeys relative to the preinoculation levels. Although the
peak (24 h) IFN-␣ mRNA levels tended to be marginally higher in
the untreated monkeys, the difference was not significant. In the
oseltamivir-treated and untreated groups of animals, IFN-␣ mRNA
levels declined to baseline by day 7 PI (Fig. 7). Furthermore, the
mRNA expression levels for three of the four IFN-inducible genes,
OAS, IFN-␥, and IP-10, were not significantly different in the oseltamivir-treated and untreated groups, although a trend toward
increased expression of IP-10 in the untreated animals compared
with the oseltamivir-treated animals was apparent (Fig. 7). Thus,
despite the significant reduction in viral replication in oseltamivirtreated monkeys, both the treated and untreated monkeys demonstrated a similar gene expression pattern for IFN-␣, IFN-␥, and
most ISGs (Fig. 7).
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Days PI
The Journal of Immunology
Although the mRNA levels of the genes assessed were much
lower than those in animals inoculated with live influenza virus,
there was a low level induction of IFN-␣, OAS, IP-10, and MxA
in the animals inoculated with heat-inactivated virus (Fig. 7).
There was no change in gene expression in the tracheas of the
oseltamivir-only treatment group (Fig. 7).
In marked contrast to the other ISGs measured, the pattern of
MxA mRNA expression in the trachea was very different. The
level of MxA mRNA was significantly higher in the oseltamivirtreated animals than in the untreated animals ( p ⬍ 0.05), the heatkilled virus animals ( p ⬍ 0.01), or the oseltamivir sham animals
( p ⬍ 0.001) (Fig. 7E). Thus, IFN-␣ levels were relatively similar
in treated and untreated animals, but MxA mRNA expression was
significantly increased in the tracheas of animals in which viral
replication was suppressed by oseltamivir treatment. Taken together, these results suggest that there is active suppression of
MxA by a viral protein that is produced during influenza virus
replication.
In mice, an intact Mx gene confers resistance to experimental influenza virus infections. Haller et al. demonstrated that in the presence of IFN, mice with a functional Mx1 gene, the murine homologue of the human MxA gene, were more likely to survive a lethal
influenza virus challenge than mice without a functional Mx1 gene
(3). IFN-␣ is required to induce the Mx1-mediated influenza virus
resistance, because treatment of mice with an anti-IFN Ab eliminates the beneficial effect of the intact Mx1 gene (3, 24, 25). However, the role of IFN-induced MxA in controlling influenza virus
replication in humans and other animals is unclear. Treatment with
oseltamivir before influenza inoculation reduces peak viral replication in animal models (26, 27) and humans (11) and, thus, oseltamivir provides a straightforward approach to explore the effect
of viral replication on antiviral immunity. In the current study we
found that reduced viral replication in the tracheas of oseltamivirtreated animals did not affect the expression levels of most molecules in the IFN-␣␤ pathway. As in influenza virus infection of
humans and other animal models (28 –30), the untreated monkeys
had dramatically increased mRNA levels for these gene products
by 24 h PI. However, the mRNA level of MxA was significantly
lower in the tracheal lavages of untreated monkeys compared with
those of the oseltamivir-treated monkeys. The simplest explanation
for these observations is that high-level virus replication specifically blocks transcription of the MxA gene. This inverse in vivo
relationship between the level of influenza virus replication and the
level of MxA mRNA expression has not been previously described. Such a relationship suggests that to achieve maximal replication in rhesus monkeys and presumably humans, influenza virus suppresses MxA expression.
Although there was more cytopathology in the sloughed cells in
the tracheal lavages from untreated monkeys than from treated
monkeys, for almost all genes tested the mRNA expression patterns in treated and untreated monkeys were very similar. Thus, in
both untreated and treated monkeys all ISGs measured were markedly elevated, with the significant exception of the key antiinfluenza effector molecule MxA. Thus, in the tracheas of oseltamivir-treated animals, viral replication was significantly reduced
and MxA expression was significantly increased in comparison to
the MxA expression levels in untreated monkeys. Of note, while
IFN-␣ expression was apparently required for MxA expression,
viral replication was not required for IFN-␣ to be produced, as the
heat-killed influenza virus induced weak IFN-␣ and MxA responses in tracheas. Finally, oseltamivir treatment did not directly
induce MxA expression in rhesus monkeys, ruling out the pharmacologic induction of MxA.
Although the above observations suggest that unsuppressed
MxA expression could significantly blunt influenza virus replication in primates, the mechanism by which MxA controls influenza
virus replication in human cells is unknown. In mouse cells, Mx1is
expressed in the nucleus and specifically blocks the replication of
orthomyxoviruses (24). Because of its location, Mx1 inhibits the
primary transcription of influenza virus mRNA in the host cell
nucleus (31). In contrast, human MxA localizes to the smooth
endoplasmic reticulum in the cytoplasm of human cells (32) and,
thus, MxA would not be expected to affect the primary transcription of influenza virus mRNA. When murine 3T3 cells line are
engineered to constitutively express high levels of MxA and are
infected with influenza virus, viral mRNAs are transcribed normally by the virion-associated RNA polymerase in the nucleus.
Viral protein synthesis and genome amplification is strongly inhibited in these cells, although viral mRNAs direct viral protein
synthesis in vitro and appear to be efficiently transported to the cell
cytoplasm. These results suggest that, in mouse 3T3 cells, MxA
interferes with either intracytoplasmic transport of viral mRNAs,
viral protein synthesis, or translocation of newly synthesized viral
proteins to the cell nucleus (33). More recent studies have shown
that if MxA is artificially made to translocate into the nucleus of
3T3 cells, it binds newly synthesized influenza virus nucleoprotein, preventing viral transcription (4, 31, 33). There is only one
report of MxA-mediated antiviral activity in human cells; it has
been demonstrated that MxA inhibits cytoplasmic LaCrosse virus
genome replication (34). However, to our knowledge IFN-induced
MxA-mediated suppression of influenza virus replication in human
cells has never been reported.
The transcription of MxA has been studied and it is known that
the regulation of MxA expression is tightly controlled and, in human cell lines, is dependent on the expression of IFN-␣␤ or IFN-␶
(5). Induction of IFN-␣␤ occurs after TLRs, the retinoic acidinducible gene I (RIG-I), or the melanoma differentiation-associated gene 5 (MDA-5) bind dsRNA or ssRNA, leading to the
activation of IFN-regulatory factors 3 and 7 (IRF-3 and IRF-7),
NF-␬B, and AP-1 (35–37). Secreted IFN-␣␤ binding to the IFN-␣
receptor activates ISG factor 3 (ISGF3), which binds the ISRE (3,
24, 35). ISGF3 binding to ISRE activates the transcription of MxA.
Although activated IRF-3 and IRF-7 can form complexes with
p300 and CREB-binding protein (CBP) that induce transcription of
a subset of ISREs independently of IFN-␣␤ (38), MxA transcription does not occur by this mechanism. Further, in vivo MxA
mRNA expression appears to lack a strong IFN-␣␤ independent
response; thus, this mode of gene regulation does not seem to play
a significant role in inducing MxA expression (39). In fact, MxA
expression and MxA-mediated resistance to influenza virus is associated with IFN-␣␤ -dependent ISRE induction (5). It has recently been shown that, in influenza-infected human cells, type I
and type III IFN activity is required for MxA expression (5). Thus,
the suppression of MxA in the setting of increased IFN-␣ expression, as seen in the influenza-infected macaques, must be mediated
through inhibition of the specific transcriptional regulators that
drive MxA expression.
Although we do not know the mechanism by which high level
influenza virus replication interacts with the transcriptional regulators of MxA expression in vivo, there are examples of viral proteins that directly bind to ISREs and regulate the expression of
specific ISGs. Thus, the core protein of hepatitis C virus can directly activate OAS expression, presumably to moderate viral replication (40), and the precore and core proteins of hepatitis B virus
directly down-regulate MxA expression by binding to both of the
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Discussion
2393
2394
macaques are well suited for the detailed studies of influenza virus
pathogenesis and immunity (13, 14) that are needed to produce
novel and clinically useful interventions for seasonal and pandemic influenza.
As in humans (11), oseltamivir prophylaxis did not alter the
timing or strength of HI Ab responses in rhesus monkeys, but there
was a trend toward fewer influenza-specific T cells in the PBMCs
of oseltamivir-treated animals compared with untreated animals.
Of note, even the monkeys inoculated mucosally with heat-killed
virus made innate antiviral and anti-influenza plasma Ab responses. The Ab responses were relatively weak and delayed compared with the responses in animals inoculated with live virus, but
it is clear that mucosal exposure to moderate amounts of inactivated virus can elicit adaptive anti-influenza immune responses.
The relatively low expression levels of IFN-␣ and the ISGs in
response to the killed virus exposure may contribute to these weak
Ab responses.
This is the first report showing that high level influenza replication interferes with the innate antiviral immune responses in primates by actively suppressing expression of MxA. The most direct
explanation for these findings is that high-level influenza virus
replication results in production of a viral protein or proteins that
block MxA expression and that this may permit the virus replication to continue despite an otherwise strong innate antiviral immune response. Additional experiments are needed to determine
which influenza protein depresses MxA transcription in the respiratory epithelia where viral replication is occurring. However, the
results reported here highlight the complex and dynamic relationship between influenza A virus replication and the innate antiviral
immune response of primates.
Acknowledgments
We thank Richard Webby and David Walker at St. Jude Children’s Research Hospital for the influenza A virus and tissue culture protocols. The
Primate Services Unit at California National Primate Research Center, Jun
Li, and Eleonora Sanchez provided excellent technical assistance.
Disclosures
The authors have no financial conflict of interest.
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ISREs that control IFN-induced expression of MxA (41). Hepatitis
B virus-mediated regulation of MxA expression appears to be at
work in vivo as well, because chronically infected hepatitis B virus
patients with high levels of viral replication have low levels of
IFN-induced MxA, while other ISGs such as OAS are expressed at
high levels (41). Thus, the viral core or capsid proteins of two
hepatitis viruses control ISG expression by binding to ISREs and
either enhance or suppress ISG expression, depending on the specific interaction. Based on the above precedents with two hepatitis
viruses, it is likely that high-level influenza replication suppresses
MxA expression by a specific viral protein, such as the nucleocapsid protein, that binds to one or more of the ISREs. However, the
viral molecule and mechanism responsible for influenza A virusmediated suppression of MxA mRNA expression remains to be
determined.
Nonhuman primates were chosen for these studies because profound differences exist between the immune systems of mice and
humans (42, 43) and the laboratory mouse strains commonly used
in influenza research do not express Mx1. Because Mx1 is a key
mediator of the innate immune responses to influenza in wild mice,
the lack of Mx1 expression in laboratory mice fundamentally alters
the host virus relationship in ways that affect the adaptation of
influenza virus to mice, the levels of viral replication, influenza
virus pathogenesis, and the nature of the anti-influenza virus immune response. Further, the influenza viruses responsible for human seasonal influenza are highly adapted to the human host and,
in general, are not easily transmitted to and have aberrant pathogenicity in nonhuman hosts (44). Serial passage of some human
influenza virus isolates in eggs and small animals can result in
adaptation of the virus to replicate in naturally nonsusceptible
hosts. Thus, the features of transmission and pathogenesis unique
to human influenza virus infection are lost in animal models that
use host-adapted viruses (45–50). These serial passage strategies
exert extreme selection pressure on human influenza virus isolates
to adapt to replication in an abnormal environment producing altered pathogenesis. Thus, the mouse-adapted strain of influenza A
virus (PR8) is highly virulent and often fatal in mice, but seasonal
influenza is rarely fatal in humans. This feature of influenza virus
pathogenesis was also used to reduce the virulence of human influenza strains and produce a safe live influenza vaccine. The currently licensed, live-attenuated, cold-adapted influenza vaccine
uses a master donor virus that is adapted for replication in primary
chicken kidney cells at low temperature, producing a virus capable
of only limited nonpathogenic infection of humans and limited
transmission among humans (51). Ferrets are more susceptible to
infection with human influenza isolates than mice and develop
rhinitis that is similar to that in humans. However, the ability of
human isolates to readily infect ferrets varies with influenza strains
(50). In addition, differences in the immune systems of primates
and carnivores and the lack of reagents to study immune responses
in ferrets limit their role as models of human anti-influenza immunity (52–54). Macaque monkeys are naturally infected with human strains of influenza virus as evidenced by the presence of HI
Abs to circulating human isolates in captive animals (Table II and
Ref. 55), and macaques can be experimentally infected with human influenza virus isolates with varying degrees of virulence
(56 –58). Furthermore, as previously reported and confirmed here,
the level and kinetics of viral replication and the timing and nature
of the antiviral immune response in experimentally infected humans (28, 59, 60) and macaques (56 –58) are very similar. The
relatively close phylogenic relationship between humans and macaques provides a high degree of homology between the immune
systems of these species, and many of the reagents used to study
human immune responses also work in rhesus macaques. Thus,
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