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J Virol, February 1998, p. 1704-1708, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Coadministration of DNA Encoding Interleukin-6 and Hemagglutinin
Confers Protection from Influenza Virus Challenge in Mice
Diane L.
Larsen,1
Naomi
Dybdahl-Sissoko,1
Martha W.
McGregor,1
Robert
Drape,2
Veronica
Neumann,2
William F.
Swain,2
D. Paul
Lunn,3 and
Christopher W.
Olsen1,*
Department of Pathobiological
Sciences1 and
Department of Medical
Sciences, School of Veterinary Medicine, University of
Wisconsin
Madison, Madison, Wisconsin
53706,3 and
PowderJect Vaccines, Inc.,
Madison, Wisconsin 537112
Received 21 July 1997/Accepted 21 October 1997
 |
ABSTRACT |
This study was conducted to investigate whether Accell gene gun
coadministration of DNA encoding human interleukin-6 (IL-6) would
enhance protective immune responses in mice to an equine influenza A
virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone
exhibited accelerated clearance of homologous challenge virus but were
not protected from infection. In contrast, mice that received both HA
and IL-6 DNA had no detectable virus in their lungs after challenge.
These results strongly support the use of IL-6 as a cytokine adjuvant
in DNA vaccination.
| |
TEXT |
Influenza A viruses are important
pathogens in a variety of mammalian and avian species (20).
In humans, this virus is one of the most important respiratory
pathogens, with infection leading to extensive morbidity and greater
than 20,000 deaths in the United States during epidemic years
(2). In horses, influenza A virus infection is also a
medically and economically important disease throughout the world. In
particular, it is one of the most common causes of viral respiratory
disease among horses in North America (19, 36). As with
humans, because of the high morbidity and economic losses associated
with outbreaks, intense vaccination programs are employed for horses in
an effort to control infection with influenza virus. Recovery from
natural infection in horses results in complete immunity to reinfection
for at least 6 months and partial immunity for over 1 year
(10). However, the inactivated, whole-virus vaccines that
are commercially available for horses offer only limited
short-term protection (19). Therefore, new vaccines
that elicit responses more like the responses to natural infection are needed.
We have evaluated Accell (PowderJect Vaccines Inc., Madison, Wis.) gene
gun-mediated DNA vaccination as an alternative approach to influenza
virus vaccination. DNA vaccination has been shown previously to elicit
immune responses to a wide variety of viral, bacterial, and protozoal
pathogens (7, 11, 31, 41). In particular, immune responses
to avian influenza virus infection in chickens and human influenza
virus infection in mice and ferrets have been demonstrated following
DNA administration via intravenous, intramuscular, intranasal, and gene
gun-mediated routes of delivery (8, 37, 38, 40). Cutaneous
administration of DNA with the gene gun is, however, the most efficient
approach, requiring 250 to 5,000-fold less DNA than parenteral
injection techniques (9, 24). This technique also provides
an added safety advantage over intramuscular injection since the
administered DNA can be removed from the body through normal epidermal
cell turnover (33).
Compared to administration of preformed protein antigen, DNA
vaccination is particularly attractive for several reasons. Active synthesis of the immunogen de novo in transfected cells facilitates antigen expression in native form and expression by major
histocompatibility complex class I as well as class II molecules
(11, 40). In addition, DNA vaccination appears to be capable
of generating long-term humoral and cellular immune responses
(43) and may provide better heterologous protection against
antigenic-drift variants of influenza virus (40). As such,
this approach may be superior to the currently available killed equine
influenza virus vaccines. We have demonstrated previously that Accell
gene gun-mediated DNA vaccination with the hemagglutinin (HA) gene of
A/Equine/Kentucky/1/81 (H3N8) (Eq/KY) virus induces virus-specific antibodies (Abs), including virus-neutralizing (VN) Abs, in mice (23). However, only partial protection from challenge
infection was achieved with this HA DNA vaccine unless a very prolonged time period was allowed between doses of DNA. In contrast, recovery of
mice from a previous infection with Eq/KY virus conferred complete immunity to homologous virus challenge 6 weeks later (23).
We hypothesized that addition of a cytokine adjuvant could enhance the
immune responses generated by our HA DNA vaccine and subsequent protection from infection, thus mimicking the host response to natural
infection. Previous studies have investigated the use of a wide variety
of cytokines (interleukin-1 [IL-1] to IL-8, IL-12, interferon,
granulocyte-macrophage colony-stimulating factor, and tumor necrosis
factor) as vaccine adjuvants (4, 12, 15, 16, 21, 25-27,
42), but our study is unique in its use of IL-6 DNA as a vaccine
adjuvant administered by gene gun delivery. IL-6 is a critical factor
in end-stage differentiation of B cells into immunoglobulin A
(IgA)-secreting plasma cells (13, 17), and studies by Ramsay
et al. of IL-6 knockout mice demonstrated that IL-6 is vital for
maintenance of mucosal IgA responses (26). However, IL-6
also stimulates proliferation of T cells (39). Immunity to
influenza is thought to be similarly dependent upon both local IgA
responses for protection at the mucosal surfaces and cellular immune
responses for clearance of virus from the body (20). Thus,
we hypothesized that coadministration of IL-6 DNA would enhance the
level of protection elicited by an influenza virus DNA vaccine.
Effect of HA DNA vaccination, with or without IL-6 DNA as a
cytokine adjuvant, on protection from homologous virus challenge
infection.
DNA vaccination was conducted with the Accell gene gun
(11). Two doses of 5.0 µg of total DNA were administered
into the epidermis of BALB/c mice, with a 3-week interval between
vaccinations. The Eq/KY HA gene had been cloned previously
(23) into a cytomegalovirus (CMV) promoter-based eukaryotic
expression vector (pWRG7077; kindly provided by James Fuller,
PowderJect) containing intron A from CMV, a poly(A) signal, and the
kanamycin resistance gene. The resulting plasmid is hereafter
designated pWRGHA. HA protein expression from pWRGHA was confirmed by
immunofluorescent-antibody staining of transiently transfected
Madin-Darby canine kidney (MDCK) cells (23). The human IL-6
(HuIL-6) gene was also expressed in a CMV promoter-based plasmid
(32) and is hereafter referred to as pWRGIL-6. IL-6
expression from pWRGIL-6 was confirmed by testing the supernatants of
transiently transfected MDCK cells with both a commercially available
enzyme-linked immunosorbent assay (ELISA) kit (see below) and the
B9-cell assay for IL-6 bioactivity (1).
In our first experiment, the mice were divided into three vaccination
groups. One group of mice (n = 10) received only
pWRG7077 DNA and served as controls. The second group of mice
(n = 20) received 2.5 µg of pWRGHA DNA plus 2.5 µg
of pWRG7077 control DNA, and the third group of mice
(n = 20) received 2.5 µg of pWRGHA DNA
plus 2.5 µg of pWRGIL-6 DNA. The reason for including control pWRG7077 DNA in the HA group was to equilibrate the total amount of DNA given and to account for any promoter competition in the mice
receiving HA plus IL-6 DNA. However, for the sake of clarity, this
HA-pWRG combination is hereafter referred to simply as HA DNA. To
confirm our initial protection-from-challenge results and to obtain
samples for assessment of mucosal immune responses, a second round of
similar experiments was conducted (n = 10 mice per
group), this time including a fourth group of mice that received pWRGIL-6 DNA plus pWRG7077 DNA. This group was included as an additional control to rule out any direct protective effect of IL-6 in
the absence of HA antigen expression. Two weeks after the second
vaccinations, all mice in the first experiment and half of the mice in
each vaccination group in the second experiment were challenge infected
with 6.65 × 104 50% egg infectious doses
(EID50s) of Eq/KY virus by intranasal instillation under
light methoxyflurane (Metofane; Pittman Moore, Mundelein, Ill.)
sedation. (The Eq/KY virus was obtained from the Influenza Virus
Repository at the University of WisconsinMadison and was propagated
in embryonated chicken eggs [22].) The remaining mice
in the second experiment were euthanatized 2 weeks after their second
vaccination to obtain nasal wash samples for assessment of mucosal
immune responses in the absence of a challenge infection. Challenged
mice were euthanatized either 3 or 5 days after infection in experiment
1. (Half of the mice in each group were euthanatized on each day.) All
challenged mice in experiment 2 were euthanatized 3 days after
infection.
To assess protection from infection, the levels of virus in the lungs
of challenged mice were calculated in EID
50s/gram of
lung
tissue as previously described (
23,
28). Virus titers
for
vaccination groups were compared statistically by one-way
analysis of
variance (ANOVA) with pairwise contrasts. The titers
of virus in the
lungs of the mice from the first experiment are
shown in Fig.
1A. Compared to the control vaccinated
mice, the
mice that received HA DNA had reduced levels of virus in
their
lungs by day 3 after challenge (
P = 0.001); they
had also cleared
their infections by 5 days after challenge. However,
the mice
vaccinated with HA plus IL-6 DNA were completely protected
from
pulmonary infection, as evidenced by a lack of detectable virus
in
their lungs after challenge. The difference in virus titers
between the
HA and HA plus IL-6 DNA-vaccinated mice 3 days after
challenge is
highly statistically significant (
P < 0.0001). These
results were confirmed in the second experiment (Fig.
1B). Once
again,
the mice that received both HA and IL-6 DNA were completely
protected.
The virus titers for the mice that received IL-6 DNA
without HA DNA
were comparable to those in the control DNA-vaccinated
mice, thus
confirming that the protection we observed cannot be
attributed to an
effect of IL-6 alone.
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FIG. 1.
The mean titers, and standard errors of the means,
of virus in the lungs of mice following vaccination with control
pWRG7077 DNA ( ), pWRGHA plus pWRG7077 DNA ( ), pWRGHA plus
pWRGIL-6 DNA ( ), or pWRGIL-6 plus pWRG7077 DNA ( ). Mice were
challenged with 6.65 × 104 EID50s of
Eq/KY virus by intranasal instillation under light methoxyflurane
(Pittman Moore) sedation, 2 weeks after the second vaccinations. (A)
Results from experiment 1. (B) Results from experiment 2. Virus was
detected by inoculation (in triplicate) of serial dilutions of each of
the mouse lungs into the allantoic cavities of 10-day-old embryonated
chicken eggs (23). Mice were euthanatized for collection of
lung samples either 3 or 5 days after challenge (p.i.). *, the
reduction in titer achieved by administration of HA DNA compared to
controls is statistically significant (P = 0.001)
(one-way ANOVA and pairwise contrasts); **, the reduction in titer
achieved by coadministration of HA plus IL-6 DNA compared to
administration of HA DNA alone is highly statistically significant
(P < 0.0001) (one-way ANOVA and pairwise contrasts).
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|
Measurement of serum IL-6 levels after vaccination.
HuIL-6 is
known to bind to murine IL-6 (MuIL-6) receptors and to be functional in
the mouse (39). The use of HuIL-6 DNA in this study allowed
us to distinguish between serum IL-6 activity expressed from our DNA
construct and endogenous MuIL-6. Levels of both HuIL-6 and MuIL-6 in
serum samples obtained 44 h after the first vaccinations were
determined with commercially available ELISA kits (R&D Systems Inc.,
Minneapolis, Minn.). This time period after DNA administration was
chosen based upon previous results of gene gun-mediated administration
of IL-6 DNA in mice (32). Following vaccination, the level
of HuIL-6 in serum was 40 pg/ml in the mice that received IL-6 DNA but
was below the level of detection of the assay (<3 pg/ml) in the mice
that received control or HA DNA alone. The MuIL-6 levels remained below
the level of detection of the assay (<15.6 pg/ml) in all samples,
suggesting that expression of HuIL-6 by DNA administration did not
up-regulate endogenous MuIL-6 expression.
Virus-specific serum IgG, IgA, and VN-Ab production.
Virus-specific Abs were measured by isotype-specific ELISA and VN-Ab
assay (23) with serum samples obtained immediately prior to
the first vaccinations, immediately prior to the second vaccinations (3 weeks), immediately prior to challenge (5 weeks), and at the time of
euthanasia. Due to volume restrictions, blood samples from all of the
mice in each vaccination group were pooled at the time of collection.
Administration of either HA or HA plus IL-6 DNA induced Eq/KY
virus-specific serum IgG and VN Abs and primed the mice for production
of virus-specific serum IgA following challenge. However, despite the
dramatic enhancement of protection from challenge that we observed in
the mice that received HA plus IL-6 DNA, we did not observe large
differences in their immune responses compared to those in the mice
that received HA DNA alone. Virus-specific serum IgG and IgA responses
at the time of challenge were identical in both groups of mice (Fig.
2A and B), and only subtle enhancements
in titer (1 serum dilution factor) were observed after challenge in the
mice that received HA plus IL-6 DNA. The kinetics of the VN-Ab
responses (Fig. 2C) paralleled those of the serum IgG responses. At the
time of challenge, the VN-Ab titer for the mice that received HA plus
IL-6 DNA was 50% higher than the titer for the mice that received HA
DNA alone. However, this difference represents less than 1 serum
dilution factor, and it is very unlikely that this small difference
could account for their distinctly enhanced protection from pulmonary
infection.
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FIG. 2.
(A and B) Virus-specific serum IgG (A) and IgA (B)
titers, as measured by ELISA, in mice vaccinated with control pWRG7077
DNA, pWRGHA plus pWRG7077 DNA (), or pWRGHA plus pWRGIL-6 DNA ().
The ELISAs were performed as previously described (23).
ELISA titers are defined as the reciprocal of the highest dilution of
serum for which the optical density was a least twice as great as the
optical density of the negative control sample on that plate. (C) VN-Ab
titers in mice vaccinated with control pWRG7077 DNA, pWRGHA plus
pWRG7077 DNA (), or pWRGHA plus pWRGIL-6 DNA (). VN Abs were
measured by inoculation (in triplicate) of MDCK cells with serial
dilutions of serum incubated with 50% tissue culture infective doses
(50 doses) of Eq/KY virus (23). The VN-Ab titers are defined
as the reciprocal of the highest dilution of serum that completely
inhibited the Eq/KY virus-induced cytopathic effect. In each panel, the
times of sampling are shown. pre, immediately prior to the first
vaccination; 3wk, immediately prior to the second vaccination; 5wk,
immediately prior to challenge; and 3dp.i. or 5dp.i., 3 or 5 days after
challenge infection.
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Virus-specific mucosal IgG and IgA responses.
In the second
experiment, nasal wash samples were collected for assessment of Ab
responses in the nasal passages. These samples were obtained 2 weeks
after the second vaccination from mice that were not challenged and 3 days after infection from the challenged mice. Nasal washes were
performed after euthanasia by inserting a 22-gauge intravenous catheter
retrograde from the tracheal bifurcation toward the head, positioning
the end of the catheter at the caudal area of the nasal turbinates. One
milliliter of sterile phosphate-buffered saline plus 1% bovine serum
albumin was flushed through the catheter and collected as it drained
from the nares into a sterile petri dish. The flush was repeated three
times with the same volume of fluid. As an additional measure of
mucosal immune responses, virus-specific Abs in fecal pellets that were
collected immediately prior to challenge and at the time of euthanasia
were measured. One fecal pellet was collected per mouse and homogenized
to a concentration of 1 mg/ml (wt/vol) in phosphate-buffered saline containing 5% fetal bovine serum and 0.01% Tween 20.
No virus-specific IgA was detectable in any of the nasal washes or
fecal pellets tested. This was not simply a technical problem
with the
assay, because we could detect virus-specific IgA when
we spiked a
portion of these samples with immune sera (data not
shown).
Virus-specific IgG was detected in the nasal washes. In
fact, nasal IgG
was the only immunologic parameter that differed
substantially at the
time of challenge for the mice that received
HA plus IL-6 DNA and those
that received HA DNA alone. This may
be an important correlate to
protection against influenza virus
infection in our model system.
Virus-specific IgG was present
in the nasal washes prior to challenge
in the mice vaccinated
with HA plus IL-6 DNA, but it was not detectable
until after challenge
infection in the mice that received HA DNA alone.
In addition,
by 3 days after challenge, the virus-specific IgG titer
was fourfold
higher in the mice vaccinated with HA plus IL-6 DNA (Fig.
3).
The procedure for obtaining nasal
wash samples is very nontraumatic.
Therefore, we are confident that
this IgG does not simply represent
blood contamination at the time of
sampling.
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FIG. 3.
Virus-specific IgG titers, as measured by ELISA, in
nasal wash specimens from mice vaccinated with control pWRG7077 DNA,
pWRGHA plus pWRG7077 DNA (), pWRGHA plus pWRGIL-6 DNA (), or
pWRGIL-6 plus pWRG7077 DNA. Titers are defined as the reciprocal of the
highest dilution of sample for which the optical density was a least
twice the optical density of the negative control samples on that
plate. The times of sampling are shown. 5wk, immediately prior to
challenge; 3dp.i., 3 days after challenge infection.
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IgA is the predominant isotype in mucosal secretions because it is
selectively transported across mucosal surfaces by the
polymeric
immunoglobulin receptor transport system (
18). In
contrast,
IgG cannot utilize this transport system and, therefore,
most likely
enters mucosal secretions either via transudation
from systemic sources
or following local production. Results of
numerous studies indicate
that protection from initial infection
with influenza virus in the
upper airways of mice (
3,
14,
29,
30,
34,
35) and humans
(
5,
20) correlates with
local IgA responses. However, this
was not the case in our study,
and it is important to note that there
is a precedent for protection
from influenza virus lethal challenge in
mice in the absence of
a mucosal IgA response (
6).
Furthermore, preliminary results
of DNA vaccination of horses indicate
that protective immune responses
correlate with nasal IgG responses in
the absence of IgA prior
to challenge (
16a). To more
completely understand the immunologic
basis for the IL-6 adjuvant
effect that we have observed in mice,
future studies will be needed to
determine whether virus-specific
IgG-secreting cells are present in the
upper airways of vaccinated
mice and whether their frequency correlates
with protection from
initial infection in the nasal passages, as well
as in the lungs.
In addition, it will be very important to assess the
effect of
IL-6 DNA administration on cytotoxic-T-lymphocyte responses
in
our system, since cytotoxic T lymphocytes may be very important
in
clearance of influenza virus from the body (
20).
In summary, Accell gene gun administration of DNA encoding IL-6 induced
a very significant adjuvant effect on the protection
from challenge
infection elicited by an equine influenza virus
HA DNA vaccine in mice.
We have initiated similar studies of horses
and pigs, in both of which
influenza virus is an important pathogen.
Immune responses against
influenza virus have been elicited previously
by gene gun-mediated DNA
vaccination in both of these species
(
16a,
16b), but as in
our mouse model system, HA DNA vaccination
alone has not been
sufficient to induce complete protection from
challenge. Therefore, we
have cloned the porcine (kindly provided
by Michael Murtaugh,
University of Minnesota) and equine (kindly
provided by David
Horohov, Louisiana State University) IL-6 genes
into the pWRG7077
expression vector, demonstrated that they are
functionally
expressed in vitro in bioactive forms (
13a,
31a),
and are
currently assessing the adjuvant effect of IL-6 DNA on
influenza virus
DNA vaccination in these outbred species.
| |
ACKNOWLEDGMENTS |
We thank Virginia Hinshaw (University of Wisconsin) for providing
the Eq/KY virus isolate, Brian Aldridge (University of Wisconsin) for
performing the statistical analyses, and Dennis McCabe (Agracetus) and
Michael Macklin (PowderJect) for technical assistance with the gene
gun. We also thank Jackie Katz (Centers for Disease Control and
Prevention) for advice on the nasal lavage procedure.
This work was supported in part by grants from the Companion Animal
Fund of the University of Wisconsin (C.W.O.), the Agricultural Experiment Station of the USDA (C.W.O.), and the Grayson-Jockey Club
Research Foundation (D.P.L.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathobiological Sciences, School of Veterinary Medicine, University of WisconsinMadison, 2015 Linden Dr. West, Madison, WI 53706. Phone: (608) 265-8681. Fax: (608) 263-0438. E-mail:
olsenc{at}svm.vetmed.wisc.edu.
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J Virol, February 1998, p. 1704-1708, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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