JOURNAL OF VIROLOGY, Oct. 2006, p. 9943–9950
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 20
Induction of Innate Immunity against Herpes Simplex Virus Type 2
Infection via Local Delivery of Toll-Like Receptor Ligands
Correlates with Beta Interferon Production
Navkiran Gill, Philip M. Deacon, Brian Lichty, Karen L. Mossman, and Ali A. Ashkar*
Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine,
McMaster University Health Sciences Centre, Hamilton, Ontario, Canada
Received 19 May 2006/Accepted 4 August 2006
Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide
spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demon-
strated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus
type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that
production of beta interferon (IFN-?) but not production of IFN-?, IFN-?, or tumor necrosis factor alpha
(TNF-?) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG
oligodeoxynucleotides induced significant production of IFN-? in the genital tract and provided complete
protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-? in mice treated with
ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge.
There was no correlation between levels of TNF-? or IFN-? in the genital tract and protection against IVAG
HSV-2 challenge following TLR ligand delivery. Both TNF-??/?and IFN-??/?mice were protected against
IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly
IFN-?, mediates innate protection, mice unresponsive to type I interferons (IFN-?/?R?/?mice) and mice
lacking IFN regulatory factor-3 (IRF-3?/?mice) were treated with poly(I:C) and then challenged with IVAG
HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-?/?R?/?or
IRF-3?/?mice. Local delivery of murine recombinant IFN-? protected C57BL/6 and IRF-3?/?mice against
IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-?
production and innate antiviral immunity against HSV-2.
The innate immune response represents the first line of
defense against microbial pathogens. This system is a universal
and ancient form of host defense against infection. It has the
ability to recognize pathogens via pattern recognition recep-
tors. Toll-like receptors (TLRs) are the best-characterized
family of pattern recognition receptors. TLRs recognize con-
served epitopes of various microbial components, known as
pathogen-associated molecular patterns, that are expressed on
infectious agents and are typically needed for survival (2, 7, 15,
16, 25). In general, these epitopes are common to an entire
class of pathogen without the need for clonal differentiation of
host cells (1). It is now evident that the 11 known mammalian
TLRs each recognize distinct ligands and utilize multiple over-
lapping signaling pathways which result in innate defense
against invading pathogens (7, 15, 25, 34, 36). Examples of
pathogen-associated molecular patterns include lipopolysac-
charide (LPS) (ligand for TLR4), peptidoglycan (PGN) (ligand
for TLR2), flagellin (ligand for TLR5), double-stranded RNA
(ligand for TLR3), bacterial CpG DNA (ligand for TLR9), and
profilin (ligand for TLR11). Upon ligand binding, TLRs acti-
vate signaling through the Toll/interleukin-1 domain found in
the cytoplasmic tails of these proteins. These signaling path-
ways can be divided into common (MyD88-dependent) and
specific (MyD88-independent) categories. TLR2, -5, -7, -8, -9,
and -11 signaling is MyD88 dependent, while TLR3 and -4
signaling can be mediated through both MyD88-dependent
and -independent pathways.
Recently, the role of TLRs in innate immunity has been
studied extensively and it has been shown that TLR binding by
ligands triggers the activation of transcription factors that lead
to induction of antimicrobial factors, including inflammatory
cytokines and antimicrobial peptides (15). As such, TLR li-
gands have multiple clinical uses, including the induction of
protective innate immunity, cancer therapy, improvement of
responses to vaccines, and reduction of allergic responses (33).
Recent studies have shown that TLRs are involved in innate
immunity in a variety of ways. Specifically, activation of TLR3
and -4 signaling pathways has been shown to activate a family
of genes, the type I interferons (IFNs), which are involved in
antiviral responses (32). The type I IFNs, which include IFN-?
and IFN-?, are known factors in the antiviral immune response
(18, 31). The antiviral effects of these IFNs have been explored,
and it has been demonstrated that they inhibit all stages of
herpes simplex virus (HSV) replication (31). TLR3, -4, -7, and
-9 are known to induce type I IFN responses through the
activation of IFN regulatory factors (IRFs), including IRF-3
and IRF-7 (6). In addition to its recognition by TLR3, double-
stranded RNA is recognized by the cytoplasmic RNA helicases
RIG-I and MDA5, resulting in activation of IRF-3 and pro-
duction of type I IFNs in a TLR-independent fashion (35).
* Corresponding author. Mailing address: Centre for Gene Thera-
peutics, Department of Pathology and Molecular Medicine, McMaster
University Health Sciences Centre, Hamilton L8N 3Z5, Ontario, Can-
ada. Phone: (905) 525-9140, ext. 22311. Fax: (905) 522-6750. E-mail:
Recently, we and others (3, 5, 13, 28, 29) have shown that
HSV type 2 (HSV-2) infection can be prevented by delivering
CpG DNA or poly(I:C) to the mucosal surface prior to infec-
tion. However, the mechanisms of this innate antiviral protec-
tion were not fully defined. In this study, we have investigated
the factor(s) involved in this innate antiviral response. We have
found a correlation between IFN-? induction and protection
against intravaginal (IVAG) HSV-2 infection. Our data show
that IRF-3 activation and subsequent IFN-?/? signaling are
required for poly(I:C)-induced innate protection against
IVAG HSV-2 challenge. Finally, local delivery of murine re-
combinant IFN-? (mrIFN-?) alone protected C57BL/6 (B6)
and IRF-3?/?mice against subsequent IVAG HSV-2 chal-
MATERIALS AND METHODS
Mice. Female C57BL/6 and 129SVPasCrl mice, 8 to 12 weeks old, were
purchased from Charles River Laboratory (Quebec, Canada). TNF-??/?and
IFN-??/?mice were purchased from Jackson Laboratory. Breeding pairs of mice
unresponsive to type I interferons (IFN-?/?R?/?mice) were kindly provided by
Rolf M. Zinkernagel (Zu ¨rich, Switzerland). Breeding pairs of IRF-3?/?mice
were kindly provided by Tadatsugu Taniguchi (Japan). All mice followed a
12-h-day and 12-h-night schedule and were maintained under standard temper-
ature-controlled conditions as outlined by the Canadian Council on Animal
Viruses, cells, and reagents. HSV-2 strain 333 was grown and viral titers were
determined as previously described (23). C57BL/6 murine embryonic fibroblasts
(MEFs) were grown in 10% alpha minimal essential medium. Synthetic CpG
phosphorothioate oligodeoxynucleotide (ODN) (no. 1826) was provided by Mc-
Master University’s Molecular Biology Institute (MOBIX) (Hamilton, Ontario,
Canada). LPS (L26-54), PGN, and poly(I:C) were purchased from Sigma
(Oakville, Ontario, Canada). Plasmid containing bacterial flagella (FliC) was
kindly provided by Steven B. Mizel, Wake Forest University (North Carolina).
Recombinant FliC was then purified using a His tag affinity column from Pierce
Biotechnology (Rockford, IL). Medroxyprogesterone (Depo-Provera) was pur-
chased from Upjohn (Don Mills, Ontario, Canada).
Generation of mrIFN-?. The murine IFN-? gene was amplified from murine
genomic DNA by use of primers 5?-CAGAATTCGCTTCCATCATGAACAAC
AG-3? (sense) and 5?-TGCTCGAGGTTTTGGAAGTTTCTGGTAA-3? (anti-
sense). The resulting PCR product was cut with EcoRI/XhoI and cloned into
pMT/V5-HisA (Invitrogen). S2 Drosophila melanogaster cells grown in Schnei-
der’s Drosophila media (Invitrogen) were cotransfected with either pMT/mIFN-
?/V5-His or pMT/EGFP/V5-His and pCO-BLAST (Invitrogen) by use of Cell-
fectin (Invitrogen) according to the manufacturer’s instructions. Stable
transfectants were selected using 25 mg/ml blasticidin (Invitrogen). Supernatants
were prepared by culturing stable transfectants (murine IFN-? [mIFN-?] ex-
pressing or green fluorescent protein [GFP] expressing) in serum-free Schnei-
der’s Drosophila media lacking antibiotics supplemented with 500 mM copper
sulfate to induce transgene expression. After 72 h, supernatants were collected,
filtered, and frozen. Supernatants were assayed for mIFN-? content by use of an
enzyme-linked immunosorbent assay (ELISA) for mIFN-? (PBL BioMedical).
Treatment of mice with TLR ligands or mrIFN-?. B6 and IRF-3?/?mice, 6 to
8 weeks old, were subcutaneously injected with 2 mg of progesterone per mouse.
Doses and times of TLR ligand delivery were chosen based on our previous
works (5, 29). Four days later, B6 mice were anesthetized and treated vaginally
with CpG ODN (25 ?g/mouse), poly(I:C) (100 ?g/mouse), LPS (5 ?g/mouse),
PGN (100 ?g/mouse), FliC (80 ?g/mouse), or mrIFN-? (5,000 pg/mouse). IRF-
3?/?mice were also treated with poly(I:C) (100 ?g/mouse), CpG ODN (25
?g/mouse), or mrIFN-? (5,000 pg/mouse).
Genital HSV-2 inoculation and vaginal virus titration. Six- to eight-week-old
mice were injected subcutaneously with 2 mg progesterone/mouse 4 days prior to
treatment. Twenty-four hours after treatment with different TLR ligands, mice
were anesthetized, placed on their backs, and infected IVAG with a lethal dose
of 1 ? 104PFU of HSV-2 in 10 ?l of phosphate-buffered saline (PBS) for at least
45 min while being maintained under anesthetic. Vaginal washes were collected
daily after infection (days 1 to 3) by pipetting two 30-?l doses of PBS into and out
of the vagina six to eight times. Viral titers in IVAG washes were determined by
plaque assay on monolayers of Vero cells as previously described (23). Treated
mice were also monitored daily for genital pathology and survival for up to 4
weeks. Pathology was scored on a five-point scale: 0, no infection; 1, slight
redness of external vagina; 2, swelling and redness of external vagina; 3, severe
swelling of external vagina and hair loss in the surrounding area; 4, ulceration of
vaginal tissue, redness, and swelling; 5, continued ulceration, redness, and swell-
ing and sometimes paralysis in back legs (mice were sacrificed at this point).
ELISAs for IFN-?, IFN-?, TNF-?, and IFN-?. Tumor necrosis factor alpha
(TNF-?) and IFN-? ELISAs were conducted using Quantikine murine kits from
R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions.
IFN-? and IFN-? ELISAs were conducted using PBL biomedical kits from PBL
(Piscataway, NJ). The IFN-? ELISA kit detects mouse IFN-?A, IFN-?1, IFN-
?4, IFN-?5, IFN-?6, and IFN-?9, with a detection limit of 10 pg/ml.
Bioassay for type I interferon activity. To detect the biological activity of
IFN-? in the supernatants of mrIFN-?-expressing S2 cells, we used a standard
vesicular stomatitis virus plaque reduction assay. The supernatants were serially
diluted and transferred onto MEFs plated in 12-well plates. Standard recombi-
nant IFN-? (rIFN-?) was also serially diluted in duplicate and transferred onto
the MEFs in order to quantify the results. The MEFs with supernatants were
incubated at 37°C for 24 h. The supernatants were then removed, and the cells
were infected with 200 ?l of vesicular stomatitis virus with predetermined viral
titers for 1 h. The plates were then overlaid with methylcellulose, incubated for
24 h, and then stained for plaque count. The plaque count of the rIFN-? serially
diluted samples was used to quantify the amount of rIFN-? in the S2 superna-
tants. Supernatants from GFP-expressing S2 cells were used as a control.
FIG. 1. Vaginal delivery of certain TLR ligands increases resis-
tance to IVAG HSV-2 infection in B6 mice. Six- to eight-week-old
female B6 mice were treated with CpG ODN (100 ?g/mouse, n ? 20),
poly(I:C) (100 ?g/mouse, n ? 20), PGN (100 ?g/mouse, n ? 10), FliC
(80 ?g/mouse, n ? 10), or LPS (5 ?g/mouse, n ? 20) or left untreated
24 h prior to IVAG HSV-2 challenge (1 ? 104PFU/mouse). Chal-
lenged mice were monitored daily for genital pathology, survival, and
vaginal virus titers. (a) poly(I:C)- and CpG-treated mice showed 100%
survival compared to LPS- or PGN-treated or untreated mice. FliC-
treated mice showed 50% protection. (b) Vaginal HSV-2 titers were
examined on days 1 to 3 following viral challenge. B6 mice treated with
poly(I:C) and CpG had no viral titers, while all other groups showed
significant viral titers.
9944GILL ET AL.J. VIROL.
Histomorphology of the genital tract. To study the effects of TLR ligands on
vaginal tissue morphology, progesterone-treated mice received CpG ODN (25
?g/mouse), poly(I:C) (100 ?g/mouse), LPS (5 ?g/mouse), peptidoglycan (100
?g/mouse), or FliC (80 ?g/mouse). After 24 h, vaginal tissue was removed, fixed
in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 mm for
hematoxylin and eosin staining.
Statistical analysis. Statistical differences of the viral titers were determined
by analysis of variance followed by Tukey’s test. The statistical significances of
the survival rates were determined by the ?2test. A P value of ?0.05 was
considered statistically significant. An unpaired t test was used to find the sig-
nificant differences in cytokine production.
Vaginal delivery of only certain TLR ligands mediates pro-
tection against IVAG HSV-2 infection. While our previous
findings demonstrated that ligands for TLR3 and -9 conferred
protection against IVAG HSV-2 infection (5, 29), the activities
of additional TLR ligands remained to be studied. Our results
show that protection against HSV-2 is 100% effective using
poly(I:C) and CpG ODN IVAG treatments, 50% effective
using FliC, and ineffective using PGN and LPS treatments
(Fig. 1a). We also examined viral titers in the vaginal washes
following IVAG HSV-2 challenge (Fig. 1b). No virus was de-
tected in the vaginal washes from mice treated with poly(I:C)
or CpG ODN, while viral titers in the vaginal washes from mice
treated with other TLR ligands showed no significant reduc-
tion compared to titers from control mice. Thus, only stimu-
lation of TLR3 and TLR9 limits virus replication and induces
complete survival following IVAG HSV-2 challenge.
Effect of local delivery of TLR ligands on vaginal tissue.
Since we observed such a marked difference in the effects of
local delivery of TLR ligands against IVAG HSV-2 challenge,
we first examined whether the protection was a result of local
changes in the vaginal mucosa. There was a rapid proliferation
of the vaginal epithelium in all treatment groups, except mice
treated with LPS, compared to results with naı ¨ve mice (Fig. 2).
Treatments with CpG (Fig. 2b), PGN (Fig. 2d), and FliC (Fig.
2e) also resulted in neutrophil infiltration to the epithelium
and vaginal lumen. Treatment with poly(I:C) (Fig. 2c) caused
little or no inflammation compared to CpG ODN treatment,
which showed the highest level of inflammation in the submu-
cosa. Thickening of the vaginal epithelium and induction of
inflammatory cells, such as neutrophils, did not correlate with
TLR-induced innate protection against IVAG HSV-2 chal-
Mucosal delivery of TLR ligands induced the production of
IFN-? and/or TNF-? but not that of IFN-? or IFN-?. We and
others have reported that some TLR ligands induce innate
protection against HSV-2 (1, 5, 29); however, there has been
no report on the main factor(s) responsible for this protection.
To identify the factor(s) that plays a role in innate immunity,
we examined the cytokine environment in vivo. We examined
the production of IFN-?, IFN-?, IFN-?, and TNF-? in vaginal
washes following mucosal delivery of TLR ligands. There was
no detectable level of IFN-? or IFN-? in the vaginal washes of
these mice (Fig. 3). TNF-? was induced at either 12 or 24 h
following IVAG treatment with all TLR ligands (Fig. 3a). In
particular, TFN-? was elevated at both 12 and 24 h posttreat-
ment with CpG ODN, poly(I:C), and PGN, while it was de-
tectable only at 12, but not at 24 h, posttreatment with FliC and
LPS. Poly(I:C) treatment showed the highest level of IFN-? in
the vaginal washes, followed by CpG ODN treatment (Fig. 3b).
Treatment with other TLR ligands, however, had no or very
low levels of detectable IFN-?.
FIG. 2. Effects of TLR ligands on morphology of genital mucosa. Six- to eight-week-old female B6 mice were treated with progesterone and,
4 days later, received CpG ODN (100 ?g/mouse), poly(I:C) (100 ?g/mouse), PGN (100 ?g/mouse), FliC (80 ?g/mouse), LPS (5 ?g/mouse), or PBS
(control). Twenty-four hours later, vaginal tissues were isolated and processed for histological testing. Photomicrographs represent cross-sections
of vaginal tissues from mice from the various treatment groups. Magnification, ?200.
VOL. 80, 2006TLR LIGANDS AND INDUCTION OF IFN-?
Local delivery of poly(I:C) protected TNF-??/?and IFN-
??/?mice against IVAG HSV-2 challenge. We have observed
that local delivery of TLR ligands induced production of sig-
nificant amounts of TNF-?. However, there was no correlation
between induction of TNF-? and protection against IVAG
HSV-2 challenge. To confirm this, we treated TFN-??/?mice
with poly(I:C) and then challenged them with IVAG HSV-2.
Poly(I:C) provided complete protection against IVAG HSV-2
challenge in the absence of TNF-? (Fig. 4). IFN-? also plays an
important role in innate defense against IVAG HSV-2 infec-
tion (4). However, innate antiviral protection was not associ-
ated with IFN-? production. Local delivery of poly(I:C) or
CpG ODN in IFN-??/?mice also provided complete protec-
tion against IVAG HSV-2 challenge (Fig. 4). Thus, although
both TNF-? and IFN-? are induced following TLR ligand
stimulation, neither is essential for survival against IVAG
IFN-? signaling is required for TLR ligand-induced innate
protection against HSV-2 infection. In order to determine
whether type I IFNs, particularly IFN-?, play a key role in
innate protection against vaginal HSV-2 infection, mice unre-
sponsive to type I IFNs (IFN-?/?R?/?mice) or unable to
produce IFN-? (IRF-3?/?mice) were used. Treatment of IFN-
?/?R?/?mice with poly(I:C) showed high levels of IFN-?
production (Fig. 5a). However, when poly(I:C)- or CpG ODN-
treated IFN-?/?R?/?mice were challenged with HSV-2, all
IFN-?/?R?/?mice succumbed to infection; in contrast, con-
genic control 129SVPasCrl mice treated with poly(I:C) showed
100% survival (Fig. 5b). Interestingly, poly(I:C)- or CpG
ODN-treated IFN-?/?R?/?mice reached end point faster
than control untreated mice. IRF-3 is a key transcription factor
responsible for IFN-? production, particularly in nonimmune
cells, such as fibroblasts and epithelial cells. To determine if
local delivery of poly(I:C) leads to IFN-? production in the
absence of IRF-3, IRF-3?/?mice were treated with poly(I:C).
There was no detectable IFN-? in the vaginal washes from
poly(I:C)-treated IRF-3?/?mice compared to those from
C57BL/6 controls (Fig. 5a). Moreover, poly(I:C)-treated IRF-
3?/?mice were not protected against IVAG HSV-2 challenge
FIG. 3. Local delivery of certain TLR ligands in B6 mice induced
production of TNF-? and IFN-?. Six- to eight-week-old female B6
mice were treated with CpG ODN (100 ?g/mouse), poly(I:C) (100
?g/mouse), PGN (100 ?g/mouse), FliC (80 ?g/mouse), or LPS (5
?g/ml) or left untreated. Vaginal washes from five mice were collected
at 12 and 24 h posttreatment, pooled, and used to measure levels of
TNF-?, IFN-?, IFN-?, and IFN-? by use of ELISA. No IFN-? (a) or
IFN-? (b) was detected in these washes; however, all treatments
caused the production of TNF-? (a). IFN-? production (b) was seen
with CpG ODN- and poly(I:C)-treated mice, while no or very little
IFN-? was detected in the washes of PGN-, LPS-, and FliC-treated
mice. The experiments were repeated two times with similar results.
FIG. 4. Local delivery of poly(I:C) provides innate protection against IVAG HSV-2 challenge in the absence of IFN-? or TNF-?. Six- to
eight-week-old female IFN-??/?mice were treated with poly(I:C) (100 ?g/mouse, n ? 10) or CpG ODN (100 ?g/mouse, n ? 10) or left untreated
(n ? 10). Six- to eight-week-old female TNF-??/?mice were also treated with poly(I:C) (100 ?g/mouse, n ? 5) or left untreated (n ? 5).
Twenty-four hours posttreatment, mice were challenged IVAG with their lethal doses of HSV-2. Challenged mice were monitored daily for genital
pathology, survival, and vaginal virus titers. Poly(I:C)-treated IFN-??/?mice and TNF-??/?mice showed 100% and 80% survival, respectively. All
control untreated mice died within 10 days after IVAG HSV-2 infection.
9946GILL ET AL.J. VIROL.
FIG. 5. Treatment of IFN-?/?R?/?or IRF-3?/?mice with poly(I:C) does not provide protection against vaginal HSV-2 challenge. (a) Six- to
eight-week-old female IFN-?/?R?/?, 129SVPasCrl (129SV) (congenic control for IFN-?/?R?/?mice), or IRF-3?/?mice were treated with
poly(I:C) (100 ?g/mouse) or left untreated. Vaginal washes were collected at 12 and 24 h posttreatment, pooled (n ? 5), and used to measure levels
of IFN-? by use of ELISA. Poly(I:C) induced significant levels of IFN-? in IFN-?/?R?/?mice. However, IRF-3?/?mice treated with poly(I:C)
showed no detectable IFN-? in the vaginal washes. Vaginal washes from four mice were pooled for ELISA. The experiment was repeated two times
with similar results. (b) Six- to eight-week-old female IFN-?/?R?/?mice and their congenic controls were treated with poly(I:C) (100 ?g/mouse,
n ? 10) or CpG ODN (25 ?g/mouse, n ? 5) or left untreated (n ? 5) 24 h prior to IVAG HSV-2 challenge. Challenged mice were monitored
daily for genital pathology and survival. Poly(I:C)- or CpG ODN-treated 129SVPasCrl mice showed 100% survival compared to untreated
129SVPasCrl control mice. There was no protection against IVAG HSV-2 challenge in poly(I:C)- or CpG ODN-treated IFN-?/?R?/?mice
compared to results with untreated control mice. (c) Six- to eight-week-old female IRF-3?/?mice and their congenic control B6 mice were treated
with poly(I:C) (100 ?g/mouse) or CpG ODN (25 ?g/mouse) or left untreated 24 h prior to IVAG HSV-2 challenge. Challenged mice were
monitored daily for genital pathology and survival. Poly(I:C)- or CpG ODN-treated B6 mice showed 100% survival compared to untreated B6
control mice. There was no protection against IVAG HSV-2 challenge in poly(I:C)-treated IRF-3?/?mice compared to results with untreated
control mice. All IRF-3?/?mice treated with CpG ODN survived against IVAG HSV-2 challenge.
VOL. 80, 2006 TLR LIGANDS AND INDUCTION OF IFN-?
(Fig. 5c). However, local delivery of CpG ODN completely
protected IRF-3?/?mice against subsequent IVAG HSV-2
challenge (Fig. 5c). Thus, IRF-3 is an essential component of
IFN-? production and subsequent protection following IVAG
Local delivery of mrIFN-? provides protection against
IVAG HSV-2 challenge in B6 and IRF-3?/?mice. Since we
have seen a strong correlation between innate antiviral protec-
tion and IFN-? production, we then examined if local delivery
of biologically active mrIFN-? alone could provide protection
against subsequent IVAG HSV-2 challenge of B6 or IRF-3?/?
mice. Interestingly, local delivery of S2 cell supernatant, 25
?l/mouse containing 2 ? 105pg/ml of mrIFN-?, provided
strong innate antiviral immunity against subsequent IVAG
HSV-2 challenge in both B6 and IRF-3?/?mice (Fig. 6). All
B6 and IRF-3?/?mice receiving local delivery of control S2
cell supernatant succumbed to subsequent IVAG HSV-2
challenge. Thus, IFN-? is capable of providing protection
against vaginal HSV-2 infection.
TLRs have been shown to induce initial inflammatory and
immune reactions by binding to conserved epitopes of micro-
bial pathogens (25). Several TLR ligands have been shown to
induce the expression of type I IFN genes (6–9), which are
cytokines known to inhibit viral replication (18). The focus of
this paper was to determine which TLR ligand treatments
induce innate antiviral responses against HSV-2 and the mech-
anism behind this protection. Here we have clearly shown that
innate antiviral responses against HSV-2 following TLR stim-
ulation were strongly correlated with the production of IFN-?.
In this study, experiments were undertaken first to examine
the abilities of various TLR ligands to induce innate defense
against HSV-2 and second to identify the factor(s) that is
responsible for any observed protection. We and others have
reported previously that TLR3 and TLR9 ligands confer pro-
tection against HSV-2 both in vitro and in vivo (3, 5, 24,
28–30). However, the roles of ligands for TLR2 and -5 have not
been studied previously. Here we first wanted to test the abil-
ities of other TLR ligands to induce antiviral responses in vivo.
Complete protection against IVAG HSV-2 challenge was seen
only by treatment with poly(I:C) and CpG ODN, while treat-
ment with FliC gave only 50% protection against IVAG HSV-2
challenge. We did not see any protection with PGN or LPS
We have found previously that local delivery of CpG ODN
induces rapid thickening of the vaginal epithelium and signif-
icant recruitment of inflammatory cells (5). To examine if these
events occur with other TLR ligands and whether they corre-
late with protection against IVAG HSV-2 challenge, we exam-
ined the vaginal mucosa following treatment with TLR ligands.
We observed that the thickening of the vaginal epithelium
occurs in both protected and nonprotected groups. Although
local delivery of PGN or FliC induced similar changes in the
vaginal epithelium, including thickening and recruitment of
inflammatory cells such as neutrophils, these mice were not
protected against IVAG HSV-2 challenge compared to mice
treated with poly(I:C) or CpG ODN. There are reports that
depletion of neutrophils leads to higher viral load in the genital
tract (26). However, in these previous studies, anti-Gr1 anti-
body, which also depletes an important subset of IFN-produc-
ing dendritic cells, was used for neutrophil depletion. More-
over, it has been reported that susceptibility to genital HSV-2
infection is increased due to progesterone treatments or oo-
phorectomy, which are also associated with higher numbers of
neutrophils in the genital tract (11, 12, 19). Although we have
seen no correlation between thickening of the vaginal epithe-
FIG. 6. Local delivery of mrIFN-? provides innate protection against vaginal HSV-2 challenge of B6 and IRF-3?/?mice. Six- to eight-week-old
female IRF-3?/?mice (n ? 8) or control B6 mice (n ? 10) were treated with cell supernatants containing mrIFN-? (mrIFN-? sup) or mock
supernatants (mock sup) or left untreated. Twenty-four hours posttreatment, mice were challenged with IVAG HSV-2. Challenged mice were
monitored daily for genital pathology and survival. Local delivery of mrIFN-? provided B6 mice with 80% protection and IRF-3?/?mice with 75%
protection against subsequent IVAG HSV-2 challenge. Mice treated with mock supernatants showed no protection against IVAG HSV-2 challenge
compared to naı ¨ve mice.
9948GILL ET AL. J. VIROL.
lium and TLR ligand-induced innate protection against IVAG
HSV-2 challenge, the mechanism(s) behind this process is yet
to be investigated. One might speculate that TLR ligands could
induce cell proliferation in the vaginal epithelium but could
not induce innate antiviral responses.
We next sought to determine the factors mediating this
protection by investigating the innate antiviral cytokine envi-
ronment. It has been well-documented that various cytokines
from the interferon family, such as IFN-?, IFN-?, and IFN-?,
play important roles in protection against HSV-2 (4, 6, 10, 18,
20–22, 32). There have also been studies showing TNF-? as a
potent antiviral cytokine (24, 27, 30). Based on these findings,
we investigated various cytokines to determine whether any of
them could be responsible for the protection in vivo. We and
others (5, 24) have reported previously that poly(I:C), LPS,
and CpG ODN do not increase IFN-? production in RAW
264.7 cells. Our mouse model showed that treatment with TLR
ligands did not induce the production of detectable levels of
IFN-?. Treatment of IFN-??/?mice with poly(I:C) or CpG
ODN confirmed that IFN-? is not crucial for the TLR ligand-
induced innate antiviral immunity. Next, we examined the role
of IFN-? in the observed protection. Results of our study
demonstrated that local delivery of poly(I:C), LPS, PGN, CpG
ODN, and FliC did not induce detectable levels of IFN-?, as
assayed by ELISA. The detection limit of the kit was about 10
pg/ml, and it recognized six different IFN-? species. This sug-
gests that IFN-? may not be the mediating factor in this innate
protection against HSV-2.
We have reported recently that treatment of RAW 264.7
cells with TLR ligands induces the production of TNF-? (10).
Other studies have shown that TLR2 and TLR4 activation in
RAW 264.7 cells is associated with TNF-? production (17).
However, recent studies have demonstrated that neutralizing
TNF-? with anti-TNF-? antibody has no effect on the viral
replication in RAW 264.7 cells (22). Our mouse model showed
that local delivery of TLR ligands induced significant produc-
tion of TNF-? in the genital mucosa; however, this did not
correlate with protection against IVAG HSV-2 challenge. Spe-
cifically, high levels of TNF-? secretion are seen with LPS and
PGN treatment; however, there was no survival in either of
these two groups following HSV-2 challenge. Moreover, TNF-
??/?mice were protected against IVAG HSV-2 challenge
following local delivery with poly(I:C). Thus, it appears that
TNF-? induction is not responsible for the innate immune
protection seen following local delivery of TLR ligands.
The last cytokine to be examined was IFN-?. We have re-
ported previously that IFN-? is important in the protection
against HSV-2 in RAW 264.7 cells treated with interleukin-15
(10). Also, by use of gene expression models, IFN-? induction
has been shown to occur in RAW 264.7 cells treated with TLR
ligands (32). In this study, the levels of IFN-? produced di-
rectly correlated with the percentages of survival we saw in the
mice treated with different TLR ligands.
To confirm that IFN-? is critical in the innate protection
against vaginal HSV-2 challenge, we used IFN-?/?R?/?mice,
which are unresponsive to IFN-?. We found that treatment of
these mice with poly(I:C) resulted in IFN-? production; hence,
the ability to produce IFN-? is not affected. However, poly(I:
C)- or CpG ODN-treated mice did not survive vaginal HSV-2
challenge. IFN-?/?R?/?mice succumbed to infection even
more quickly than naı ¨ve wild-type congenic controls, while
wild-type poly(I:C)-treated mice showed 100% protection.
This clearly indicates the crucial role of type I IFNs (IFN-?/?)
in the innate protection against HSV-2. Although IFN-?/
?R?/?mice lack signaling for both IFN-? and IFN-?, we have
already determined that TLR ligands do not induce significant
levels of IFN-?. Therefore, we sought to attribute our findings
to the role of IFN-? in this model. In nonimmune fibroblasts
and epithelial cells, IRF-3 is an important transcription factor
in virus-mediated IFN-? production. Local delivery of
poly(I:C) in IRF-3?/?mice induced no detectable levels of
IFN-?, and the mice were not protected against subsequent
IVAG HSV-2 challenge. However, as expected, local delivery
of CpG ODN provided complete protection against IVAG
HSV-2 infection in IRF-3?/?mice. This observation suggests
that in the vaginal mucosa, nonimmune fibroblasts and/or ep-
ithelial cells are the primary producers of IFN-?, as opposed to
immune cells, which produce predominantly IFN-? and re-
quire IRF-7 for full activation (14). Finally, to confirm the key
role of IFN-? in the innate protection against IVAG HSV-2
infection, we examined whether mrIFN-? alone could protect
B6 and IRF-3?/?mice against IVAG HSV-2 challenge. Inter-
estingly, local delivery of mrIFN-? provided protection against
HSV-2 challenge. While poly(I:C) is a known ligand of TLR3,
it is likely that the cytoplasmic RNA helicases RIG-I and
MDA5 also play a role in IFN-? induction in response to this
We have demonstrated a clear correlation between IFN-?
secretion and protection from HSV-2 TLR ligands in vivo. This
correlation suggests a vital role for IFN-? in the innate pro-
tection against HSV-2 infection and supports further study in
this area. It is yet to be determined how IFN-?, following local
delivery of TLR ligands, exerts its antiviral activity against
HSV-2. From our previous work, we know that HSV-2 enters
the epithelial cells, but it is not clear at which step the viral
replication is blocked. An understanding of TLR ligands in
innate antiviral responses may provide new insights in defense
against viral infections, including HSV and human immunode-
ficiency virus type 1 infections.
We thank Steven B. Mizel for providing plasmid containing FliC,
Jonathan Bramson for providing S2 Drosophila cells, Rolf M. Zinker-
nagle and Ken L. Rosenthal for providing breeding pairs of IFN-?/
?R?/?mice, and Tadatsugu Taniguchi via Thomas Moran for provid-
ing breeding pairs of IRF-3?/?
MacKenzie, Mary Jo Smith, Mary Bruni, Jennifer Newton, Amanda
Kwant, and Amy Patrick for technical assistance.
This work was supported by grants from the Hospital for Sick Chil-
dren Foundation and the Canadian Institutes of Health Research
(CIHR) to Ali A. Ashkar. Ali A. Ashkar is a recipient of a career
award in health sciences from Rx&D/CIHR.
mice. We also thank Randy
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