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Journal of Virology, July 1999, p. 5527-5534, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Anamnestic Neutralizing Antibody Response Is
Critical for Protection of Mice from Challenge following Vaccination
with a Plasmid Encoding the Japanese Encephalitis Virus Premembrane
and Envelope Genes
Eiji
Konishi,1,2,*
Masaoki
Yamaoka,2,
Khin-Sane-Win,2
Ichiro
Kurane,3,
Kazuo
Takada,3,§ and
Peter
W.
Mason4
Department of Health Sciences, Kobe
University School of Medicine, Kobe 654-0142,1
Department of Medical Zoology, Kobe University School of
Medicine, Kobe 650-0017,2 and Department
of Microbiology, Kinki University School of Medicine, Osaka-sayama
589-8511,3 Japan, and Plum Island Animal
Disease Center, Agricultural Research Service, U.S. Department of
Agriculture, Greenport, New York 119444
Received 19 November 1998/Accepted 30 March 1999
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ABSTRACT |
For Japanese encephalitis (JE), we previously reported that
recombinant vaccine-induced protection from disease does not prevent challenge virus replication in mice. Moreover, DNA vaccines for JE can
provide protection from high challenge doses in the absence of
detectable prechallenge neutralizing antibodies. In the present study,
we evaluated the role of postchallenge immune responses in determining
the outcome of JE virus infection, using mice immunized with a plasmid,
pcDNA3JEME, encoding the JE virus premembrane (prM) and envelope (E)
coding regions. In the first experiment, 10 mice were vaccinated once
(five animals) or twice (remainder) with 100 µg of pcDNA3JEME. All of
these mice showed low (6 of 10) or undetectable (4 of 10) levels of
neutralizing antibodies. Interestingly, eight of these animals showed a
rapid rise in neutralizing antibody following challenge with 10,000 50% lethal doses of JE virus and survived for 21 days, whereas only
one of the two remaining animals survived. No unimmunized animals
exhibited a rise of neutralizing antibody or survived challenge. Levels
of JE virus-specific immunoglobulin M class antibodies were elevated
following challenge in half of the unimmunized mice and in the single
pcDNA3JEME-immunized mouse that died. In the second experiment, JE
virus-specific primary cytotoxic T-lymphocyte (CTL) activity was
detected in BALB/c mice immunized once with 100 µg of pcDNA3JEME 4 days after challenge, indicating a strong postchallenge recall of CTLs.
In the third experiment, evaluation of induction of CTLs and antibody
activity by plasmids containing portions of the prM/E cassette
demonstrated that induction of CTL responses alone were not sufficient
to prevent death. Finally, we showed that antibody obtained from
pcDNA3JEME-immunized mice 4 days following challenge could partially
protect recipient mice from lethal challenge. Taken together, these
results indicate that neutralizing antibody produced following
challenge provides the critical protective component in
pcDNA3JEME-vaccinated mice.
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INTRODUCTION |
Japanese encephalitis (JE) is a
mosquito-borne viral disease causing infection of the central nervous
system in humans and equines. It is generally believed that JE virus
present in mosquito saliva replicates at or near the bite site and is
then transported via the bloodstream into the brain, where it may cause
infection and encephalitis. Two major factors have been reported to be
important for protection from encephalitis: neutralizing antibody and
cytotoxic T lymphocytes (CTLs) specific for JE virus. Passive transfer
of monoclonal antibodies to the envelope (E) protein (1, 5, 21), T cells obtained from infected mice (23, 25), and
CTLs (26) can protect mice from a lethal challenge. High
levels of neutralizing antibody (29) and JE virus-specific T
lymphocytes (8) have been detected in JE patients in the
convalescent phase.
We have previously studied the immunogenicity of JE gene products in a
mouse model using recombinant poxviruses expressing the signal of the
premembrane (prM), the prM gene, and the envelope (E) gene. Cells
infected with these poxviruses produce subviral extracellular particles
(EPs). These subviral particles are similar to the slowly sedimenting
hemagglutinin particles produced by cells infected with JE virus,
suggesting that the prM, membrane (M), and E proteins in these EPs are
comparable to the authentic forms of these proteins (11, 13,
22). Mice immunized with poxvirus-based recombinants encoding the
signal-prM-E gene cassette induced high levels of neutralizing antibody
and memory CTLs and were protected from lethal challenge (9, 12,
14). However, these mice were not protected from infection by the
challenge virus, since high levels of antibody to the nonstructural
(NS) proteins were detected in mice surviving challenge
(11).
Recently, naked DNA plasmids encoding flavivirus genes have been
reported to induce neutralizing antibody and/or protection in mice,
using the NS1 gene of JE virus (19) and the prM/E gene of
dengue type 2 (6, 30), St. Louis encephalitis
(27), and tick-borne encephalitis (31) viruses.
We have demonstrated that mice immunized with a plasmid encoding the JE
virus signal-prM-E gene cassette (pcDNA3JEME) were also protected from
a lethal challenge (15). Interestingly, although mice
immunized with this DNA produced CTLs that could be detected after in
vitro stimulation, the levels of neutralizing antibody induced by these
DNAs were low or undetectable. Therefore, this system provides a mouse
model useful for studying the mechanism of protection against JE. Some
other DNA vaccines also have been reported to protect in the absence of
neutralizing antibody responses (19, 19a).
In this study, we analyzed the postchallenge immune responses in
pcDNA3JEME-immunized mice to elucidate the role of neutralizing antibody and CTLs in protection.
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MATERIALS AND METHODS |
Viruses.
The prototype Nakayama strain of JE virus
(20) was used for construction of plasmids, neutralization
tests, and spleen cell stimulation, and the virulent Beijing P3 strain
(22) was used for mouse challenge studies. Recombinant
vaccinia viruses used for infection of target cells in cytotoxicity
assays were vP555, encoding the prM, E, and NS1 genes of the Nakayama
strain; vP658, encoding E and NS1; vP829, encoding prM and E; and their
parent virus, vP410 (11, 22). vP829 and vP410 were also used
for preparing antigens in enzyme-linked immunosorbent assay (ELISA). A
recombinant vaccinia virus, vP857, encoding the JE virus NS1 and NS2a
genes (11) was used for immunochemical staining assays.
Plasmids.
The construction of pcDNA3JEME, a pcDNA3-based
plasmid encoding the JE virus signal sequence of prM, prM, and E genes,
has been described previously (15). In the present study,
three new pcDNA3-based plasmids encoding (i) the signal sequence of prM
and prM (nucleotides 325 to 882), (ii) the first half of E (nucleotides
883 to 1599), or (iii) the last half of E (nucleotides 1600 to 2382),
were constructed essentially by the strategy used for construction of
pcDNA3JEME (15). The JE virus cDNA encoding the signal
sequence of prM, prM, and E was amplified by PCR with a template
plasmid DNA, pARJa (containing Nakayama strain C protein cDNA sequences
fused to plasmids PM-7 and PM-6 [24]; GenBank accession no. M73710). The sense primer included an EcoRI
site, an efficient eukaryotic initiation site (16), and a
start codon, followed by the codons encoding (i)
Glu-Gly-Ser-Ile-Met-Trp of the prM signal sequence, (ii) the N-terminal
six codons of E, or (iii) codons 240 to 245 of E. The antisense primer
corresponded to (i) the C-terminal six codons of prM, (ii) codons 234 to 239 of E, or (iii) the C-terminal six codons of E, each of which was adjacent to a termination codon and an XhoI site. The
amplified cDNA was inserted into the pcDNA3 vector (Invitrogen Corp.,
San Diego, Calif.) at the EcoRI/XhoI site between
the strong eukaryotic promoter derived from human cytomegalovirus and
the polyadenylation signal derived from the bovine growth hormone. The
constructs encoding prM, the first half of E, and the last half of E
were designated pcDNA3JEprM, pcDNA3JEE1/2, and pcDNA3JEE2/2,
respectively. The proper insert of the gene cassette in these
constructs was confirmed by sequencing using a DNA sequencer (ABI 373A;
Applied Biosystems, Chiba, Japan). All plasmid DNAs (pcDNA3,
pcDNA3JEME, pcDNA3JEprM, pcDNA3JEE1/2, and pcDNA3JEE2/2) were purified
by using a Qiagen plasmid kit (Funakoshi Co. Ltd., Tokyo, Japan) as
instructed by the manufacturer and used for immunization of mice.
Mouse experiments.
Groups of five 4-week-old female ICR mice
and three 6-week-old male BALB/c mice were used for evaluating
induction of antibody and protective immunity, and groups of two
6-week-old male BALB/c mice were used for evaluating induction of CTLs.
Mice were inoculated once or twice at an interval of 2 weeks by
intramuscular injections at both thighs with 50 µl of immunogens
diluted in phosphate-buffered saline (PBS) at each site. Immunogens and
doses were pcDNA3JEME at doses of 10 and 100 µg and pcDNA3JEprM,
pcDNA3JEE1/2, pcDNA3JEE2/2, and the pcDNA3 vector at a dose of 100 µg. Two or four weeks after the last immunization, mice were
challenged by intraperitoneal (i.p.) injection with 50% lethal doses
(10,000 LD50) of the Beijing P3 strain of JE virus and
observed for 3 weeks. This challenge dose was used throughout the
study, including passive transfer experiments. Retro-orbital blood was
collected successively from each mouse 2 days before challenge and 2, 4, 6, 8, 11, 14, and 21 days after challenge unless otherwise stated.
Individual or pooled serum samples were used for evaluation of
antibody. Spleens were collected from immunized BALB/c mice just before
challenge and 4, 8, and 14 days after challenge for evaluating
induction of primary CTLs, or were collected 2 weeks after immunization for evaluating induction of memory CTLs. Spleen cell suspensions were
prepared as previously described (9) and used for
cytotoxicity assays (see below).
For passive transfer experiments, female ICR mice were immunized with
100 µg of pcDNA3JEME at 4 and 6 weeks of age and challenged with
10,000 LD50 of the Beijing P3 strain at 8 weeks of age.
Sera were collected from these mice just before challenge and 4 and 21 days after challenge. Sera were also collected from unimmunized 8-week-old female ICR mice 4 days after challenge. Immunoglobulin fractions were isolated from pooled sera by precipitation with saturated ammonium sulfate followed by extensive dialysis against PBS.
The immunoglobulin fraction corresponding to 0.2 ml of the original
serum was transferred by i.p. injection into groups of 10 unimmunized
8-week-old female ICR mice that had been challenged with 10,000 LD50 of the Beijing P3 strain 2 days earlier. These mice
were observed for 21 days for survival rates.
Neutralization tests.
Neutralizing antibodies elicited in
immunized mice were titrated as previously described (11)
except for the inclusion of complement in the virus-antibody mixture.
The neutralization titer was expressed as the highest serum dilution
yielding a 90% reduction in plaque number.
ELISA.
Antibodies to JE virus prM/E were measured by ELISA
using EPs as antigen as previously described (10). Briefly,
EPs were harvested from culture fluid of HeLa cells infected 20 h
earlier with vP829, a recombinant vaccinia virus encoding the JE virus (Nakayama strain) prM and E genes, or with vP410, the parent vaccinia virus. After precipitation with 10% polyethylene glycol, EPs were dissociated with 0.1% Triton X-100 and diluted in 0.1 M carbonate buffer (pH 9.6) for sensitization of microplates (Maxisorp; A/S Nunc,
Roskilde, Denmark). Sensitized microplates were incubated with mouse
serum samples at a 1:100 dilution, with alkaline phosphatase-conjugated anti-mouse immunoglobulin G (IgG; gamma chain specific) or IgM (mu
chain specific; EY Laboratories, San Mateo, Calif.) at a dilution of
1:1,000, and then with p-nitrophenyl phosphate (1 mg/ml).
The difference in absorbance values obtained with the recombinant and
control antigens was regarded as a measure of the antibody level
specific for prM and E. To minimize interplate variation, all values
shown were expressed relative to the reference value obtained with a
constant positive control sera, which was included in each assay plate.
Immunochemical staining assay.
Antibodies to NS1 were
titrated by immunochemical staining using vP857-infected HeLa cells as
antigen. HeLa monolayer cells grown in 96-well microplates were
infected with vP857, a recombinant vaccinia virus encoding the JE virus
NS1 and NS2a genes, at a multiplicity of infection of 2 PFU/cell. After
incubation at 37°C for 6 h, monolayers were rinsed with PBS,
fixed with ethanol, dried, and stored at 
30°C until use. For
testing, monolayers were rehydrated with PBS containing horse serum at
1% and incubated with serial twofold dilutions of test sera.
Antigen-antibody reactions were then detected with biotinylated
anti-mouse IgG (heavy and light chain specific), the ABC
(avidin-biotinylated enzyme complex) reagents, and the VIP substrate
(Vector Laboratories, Inc., Burlingame, Calif.). Titers were
represented as the maximum dilution that provided stained cells. In a
preliminary experiment using sera from unimmunized mice, cells were
occasionally stained when incubated with a 1:10 dilution of sera, but
no stained cells were observed at a 1:20 dilution. Therefore, we
decided a borderline for determination of the antibody to NS1 in mouse
sera to be positive at a dilution of 1:20.
Cytotoxicity assays.
Primary CTLs were assayed without in
vitro stimulation of spleen cells. Spleen cell suspensions prepared
from two BALB/c mice were mixed and washed three times with RPMI 1640 medium containing 10% fetal bovine serum. These cells were distributed
in triplicate in 96-well microplates at different cell densities to
provide various effector:target (E:T) ratios. The target cells used for these assays were P815 mastocytoma cells infected with vP829 or vP410
at a multiplicity of infection of 10 PFU/cell 15 to 20 h before
the assay. All target cells were labeled with
Na51CrO4, washed, and distributed evenly at
1 × 103 or 2 × 103 viable cells per
well into microplates containing effector cells. The plates were
incubated for 5 to 6 h at 37°C, and 51Cr release
into the supernatant was measured in a gamma counter. Percent specific
lysis was calculated by the following formula: 100 × (experimental release minimum release)/(maximum release minimum release), where the maximum release was obtained by lysing all
the target cells with polyoxyethylene(12)tridecyl ether (Renex; Ruger
Chemical Co., Irvington, N.J.) and the minimum release was obtained
with target cells incubated alone in RPMI 1640-10% fetal bovine serum.
Assays for secondary CTLs and the stimulation of spleen cells with the
Nakayama strain of JE virus in vitro were performed as previously
described (15). Briefly, spleen cells were stimulated by
incubation with the live JE virus antigen at 37°C for 6 days. Stimulated cells were incubated with 51Cr-labeled target
cells for 5 to 6 h at 37°C. The target cells used for these
assays were P815 mastocytoma cells infected with vP555, vP658, vP829,
or vP410.
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RESULTS |
Postchallenge antibody responses in mice immunized twice with
pcDNA3JEME.
Two groups of five ICR mice were immunized with 100 µg of pcDNA3JEME or 100 µg of the pcDNA3 vector twice at 4 and 6 weeks of age and then challenged with 10,000 LD50 of the
Beijing P3 strain of JE virus at 8 weeks of age. Prechallenge sera were
collected 2 days before challenge (defined as day 2) and examined for
neutralizing antibody. Following challenge, mice were observed for 3 weeks, and postchallenge sera were collected on days 4, 8, and 21 from all the surviving mice. The results in Table
1 show that mice immunized with
pcDNA3JEME had individual antibody titers at 1:10 to 1:20 on day 2.
After challenge, neutralization titers were elevated to 1:160 to 1:320
on day 4 and 1:320 to 1:1280 on day 8, and all mice survived for 21 days. Neutralization titers on day 21 were similar to those on day 8. Although a small number of mice were used in each experiment, the
postchallenge neutralizing antibody responses observed in the present
study were consistent with those obtained in pilot experiments (data
not shown) and in our previous study on duration of protective immunity
induced by pcDNA3JEME (15). Mice inoculated with the pcDNA3
vector did not have neutralizing antibody before challenge.
Neutralizing antibody titers did not exceed 1:10 on day 4 and 1:20 on
day 8, and all of these animals died within 10 days of challenge.
All five pcDNA3JEME-immunized mice had anti-NS1 antibody on day 21 (Table
1). Since the immunogen, pcDNA3JEME, does not contain
the NS1
gene, the presence of antibody to NS1 indicates replication
of
challenge virus in these
mice.
Postchallenge antibody responses in mice immunized once with
pcDNA3JEME.
We previously reported lower protective indices with a
single immunization with 10 or 100 µg of pcDNA3JEME than with two
immunizations (15). To analyze postchallenge antibody
responses at subprotective doses, three groups of five ICR mice
immunized once with 100 or 10 µg of pcDNA3JEME or PBS at 4 weeks of
age were challenged at 8 weeks of age. Sera were collected 2 days
before challenge and 2, 4, 6, 8, 11, 14, and 21 days after challenge
and were examined individually for neutralizing antibody (Fig. 2), IgG
(Fig. 3), and IgM (Fig. 4) antibodies to prM/E and for antibodies to
NS1 (Fig. 5).
Figure
1 shows the survival data for
these mice. Four of five mice immunized with 100 µg of pcDNA3JEME
survived, but all mice
immunized with 10 µg of pcDNA3JEME or PBS died
during the 21-day
observation period. All mice that survived for 21 days maintained
a healthy appearance throughout the observation period.
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FIG. 1.
Survival of ICR mice after challenge in the
one-immunization protocol. Three groups of five mice were inoculated
with 100 or 10 µg of pcDNA3JEME or PBS at 4 weeks of age and
challenged at 8 weeks of age.
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Differences in the kinetics of the appearance of neutralizing antibody
among these groups (Fig.
2) were
predictive of survival
(Fig.
1). Neutralization titers before challenge
were undetectable
(<1:10) in all mice but one in the pcDNA3JEME
100-µg group, which
showed a titer of 1:10 (Fig.
2). Four days after
challenge, three
of the five mice immunized with 100 µg showed
neutralization titers
of 1:80 or greater, and all three of these mice
survived for 21
days. The remaining two mice showed a neutralization
titer of
1:20 on day 4, and one of these mice died on day 16. Levels of
neutralizing antibody in the mouse that died of challenge (Fig.
2) were
1:20 or less within the survival period, whereas those
in the mouse
that survived for 21 days were 1:40 or greater on
day 8 and thereafter.
In other groups (10 µg of pcDNA3JEME and
PBS), the neutralization
titers did not exceed 1:20 throughout
the survival period and all the
mice died.
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FIG. 2.
Time course of neutralizing antibody titers in
individual mice after challenge in the one-immunization protocol. Three
groups of five ICR mice were inoculated with 100 or 10 µg of
pcDNA3JEME or PBS at 4 weeks of age and challenged at 8 weeks of age.
Sera collected 2 days before challenge and 2, 4, 6, 8, 11, 14, and 21 days after challenge were used for the test. Neutralization titers were
represented as the highest serum dilution yielding a 90% reduction in
plaque number. The symbols used in this figure are likewise used in
Fig. 3 to 5 for representing data obtained from the same mice in each
group.
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Figures
3 and
4 show the appearance of
IgG and IgM antibodies to prM/E in these mice. Overall, the appearance
of IgG antibody
(Fig.
3) followed the kinetics observed for
neutralizing antibody
(Fig.
2). Two mice immunized with 100 µg of
pcDNA3JEME showed
IgG antibody levels of over 1.2 on day 4 and
maintained these
levels until day 21 (Fig.
3). However, three other
mice in the
100-µg group showed low levels of IgG antibodies to prM/E
(less
than 0.5) on day 4 or later. Two of these three mice survived
challenge, whereas one died on day 16. All the mice immunized
with 10 µg of pcDNA3JEME or PBS alone showed ELISA levels of less
than 0.4 throughout the survival period.
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FIG. 3.
Time course of IgG antibody to prM/E in individual mice
after challenge in the one-immunization protocol. Three groups of five
ICR mice were inoculated with 100 or 10 µg of pcDNA3JEME or PBS at 4 weeks of age and challenged at 8 weeks of age. Sera collected 2 days
before challenge and 2, 4, 6, 8, 11, 14, and 21 days after challenge
were examined. Antibody levels were measured by ELISA and presented as
relative values (see Materials and Methods for details). The symbols
used in this figure are likewise used in Fig. 2, 4, and 5 for
representing data obtained from the same mice in each group.
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Time course of the appearance of IgM antibody to prM/E showed a maximum
level on day 4 in most of the mice that survived more
than 8 days after
challenge (Fig.
4). Among the 10 mice
inoculated
with 10 µg of pcDNA3JEME or PBS, which all died following
challenge,
5 showed levels of more than 0.2 on day 4, whereas all the 4 animals
which were immunized with 100 µg of pcDNA3JEME and survived
challenge
showed levels of less than 0.2. The one mouse that was
immunized
with 100 µg of pcDNA3JEME and died showed an IgM antibody
level
of more than 0.3 on day 4, suggesting that this mouse was not
effectively primed by one inoculation with 100 µg of pcDNA3JEME.
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FIG. 4.
Time course of IgM antibody to prM/E in individual mice
after challenge in the one-immunization protocol (see the legend to
Fig. 3 for details).
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Figure
5 shows the appearance of
antibodies to NS1 in the three groups of mice. The data represent the
dilution of sera that
reacted with NS1 in an immunochemical staining
assay. This assay
was based on an indirect staining protocol that
should detect
both IgG and IgM class antibodies. Since NS1 is not
included in
the vaccine DNA, the magnitude of response to NS1 can be
used
as an indicator of challenge virus replication (
11).
The antibody
to NS1 appeared on day 2 after challenge in two of the
five mice
inoculated with PBS, whereas the antibody to NS1 was first
detected
on day 4 in mice immunized with 100 or 10 µg of pcDNA3JEME.
All
mice, except one immunized with 10 µg of pcDNA3JEME that died
before antibody to NS1 was detected, showed increasing levels
of
antibody to NS1 throughout the observation period. In some
of the
animals that died within 10 days of challenge, there was
a rapid rise
in antibody to NS1 immediately preceding death (Fig.
5). The one mouse
immunized with 100 µg of pcDNA3JEME that had
a low level of
neutralizing antibody but survived showed low levels
of anti-NS1
antibody throughout the observation period, suggesting
very limited
replication of the challenge virus in this animal.
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FIG. 5.
Time course of antibody to NS1 in individual mice after
challenge in the one-immunization protocol (see the legend to Fig. 3
for details). Antibody titers were measured by an immunochemical
staining assay (see Materials and Methods for details).
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Postchallenge CTL responses.
The primary CTL responses induced
by challenge were examined in spleen cells of immunized mice. Groups of
two male BALB/c mice were immunized once with 100 µg of pcDNA3JEME at
6 weeks of age and challenged at 9 weeks of age. Spleen cells were
collected in different postchallenge days and examined for CTL activity without in vitro stimulation. In the first experiment using spleen cells obtained before challenge and on days 1, 2, and 3 after challenge, no CTL activity was detected (data not shown). In the second
experiment using spleen cells obtained before challenge and on days 4, 8, and 14 after challenge (Fig. 6),
approximately 10 to 15% of specific lysis was observed against target
cells infected with vP829 in samples obtained on day 4. Spleen cells did not lyse target cells infected with vP410. The cytotoxicity levels
obtained in mice on days 8 and 14 were lower than those obtained on day
4. In contrast, cells collected on day 0 (just before challenge) did
not lyse targets infected with vP829. These results indicate that
active CTLs were present in the immunized mice 4 days after challenge.
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FIG. 6.
Primary CTL activity in pcDNA3JEME-immunized BALB/c mice
after challenge. Four groups of two mice were immunized with 100 µg
of pcDNA3JEME at 6 weeks of age and challenged at 9 weeks of age.
Spleen cells were collected just before challenge (day 0) and 4, 8, and
14 days after challenge. Cytotoxic activities against P815 cells
infected with vP829 or vP410 were measured at E:T ratios of 400:1 and
200:1 by the standard chromium release method (see Materials and
Methods for details). vP829 is a recombinant vaccinia virus encoding
prM and E; vP410 is a parent vaccinia virus with no JE virus genes.
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Postchallenge immune responses in mice immunized with plasmids
encoding a subset of the prM/E cassette.
To further elucidate the
role of antibody and CTLs in protection, we constructed three
additional pcDNA3-based plasmids: pcDNA3JEprM, encoding the signal
sequence of prM and prM; pcDNA3JEE1/2, encoding the first half of E;
and pcDNA3JEE2/2, encoding the last half of E. Five groups of five
BALB/c mice were immunized with 100 µg of each of these plasmids,
pcDNA3JEME, or pcDNA3 at 6 and 8 weeks of age. Two of these five mice
were used to examine memory CTLs present at 10 weeks of age, and the
remaining three mice were challenged at 12 weeks of age. Figure
7 shows the cytotoxic activities of
spleen cells isolated from these groups of mice prior to challenge.
Mice immunized with pcDNA3JEE1/2 showed approximately 10 to 30% of
specific lysis against target cells expressing E (vP555, vP658, and
vP829) at E:T ratios of both 400:1 and 200:1, but no cytotoxic
activities were observed against control target cells (vP410). In mice
immunized with pcDNA3JEprM, cytotoxic levels observed against target
cells expressing prM (vP555 and vP829) were quite low, and the value
obtained with the vP829 target cells was identical to the value
obtained with vP658 target cells, which do not express prM. These data
show that memory CTLs could be detected in mice immunized with the
first half of E but not in mice immunized with prM or the second half
of E, indicating that the CTL epitope(s) is located only in the first
half of the E region.
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FIG. 7.
Memory CTLs induced in BALB/c mice immunized with
plasmids encoding the JE virus prM (prM), the first half of E (E1/2),
or the second half of E (E2/2). Mice were immunized with 100 µg of
each plasmid at 6 and 8 weeks of age. Spleen cells were collected at 10 weeks of age and stimulated by incubation with live JE virus for 6 days
at 37°C. Cytotoxic activities were measured at E:T ratios of 400:1
and 200:1 by the standard chromium release method (see Materials and
Methods for details). The target cells were P815 cells infected with a
recombinant vaccinia virus (vP555 encoding prM, E, and NS1; vP658
encoding E and NS1; or vP829 encoding prM and E) or the parent vaccinia
virus, vP410.
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Figure
8 shows the survival curve and the
course of antibody levels. Sera pooled from three mice were examined
for neutralizing
antibody and IgG and IgM antibodies to prM/E.
Consistent with
the previous results, pcDNA3JEME induced protection and
high levels
of postchallenge antibody responses, but pcDNA3 did not.
All mice
immunized with pcDNA3JEE1/2, which induced CTLs, died of
challenge.
Only low levels of neutralizing antibody (1:20 on day 4) and
the
IgG antibody to prM/E, as well as a relatively high level of IgM
antibody, were induced in these mice. pcDNA3JEprM also induced
no
protection and low levels of IgG antibody response to prM/E.
Only one
of three mice immunized with pcDNA3JEE2/2 survived, and
this animal had
a neutralization titer of 1:160 on day 21. These
results suggest that
the induction of memory CTLs to the first
half of the E region was not
sufficient to protect mice from lethal
challenge.
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FIG. 8.
Survival curves and time courses of postchallenge immune
responses in BALB/c mice immunized with plasmids encoding a subset of
the JE virus prM/E cassette. Mice were immunized with 100 µg of
pcDNA3JEprM (prM), pcDNA3JEE1/2 (E1/2), pcDNA3JEE2/2 (E2/2), pcDNA3JEME
(prM/E), or pcDNA3 (none) at 6 and 8 weeks of age and were challenged
at 12 weeks of age. Sera collected 2 day before challenge and 4, 8, and
21 days after challenge were pooled and measured for neutralizing
(NEUT) antibody and IgG and IgM antibodies to prM/E by ELISA (see
Materials and Methods for details). In a group of mice immunized with
pcDNA3JEME, one mouse died accidentally upon being given anesthesia,
and all evaluations were done with two mice.
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Passive protection by transfer of postchallenge serum.
To
clarify the role of neutralizing antibody produced following challenge
in protection, immunoglobulin fractions of the sera collected from mice
immunized by various procedures were transferred to ICR mice 2 days
following inoculation with a lethal dose of JE virus. The tested sera
included the sera pooled from pcDNA3JEME-immunized mice, sera pooled
from the same mice at 4 or 21 days postchallenge, and sera pooled from
unimmunized mice 4 days after challenge. The levels of neutralizing
antibody present in these preparations and the survival data are shown
in Table 2. Three and four of ten mice
which were given antibody obtained from immunized mice 4 and 21 days
after challenge, respectively, survived for 21 days, whereas no
protection was observed by transfer of antibody obtained from
prechallenge sera of immunized mice or postchallenge sera of
unimmunized mice. Although the levels of protection were low, we
concluded that the antibody induced a partial protection, since the
Beijing P3 strain is uniformly lethal in mice at the dose used in
this study (10,000 LD50).
View this table:
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|
TABLE 2.
Partial protection of mice passively transferred with
antibody produced in pcDNA3JEME-immunized mice after challenge
|
|
| |
DISCUSSION |
This study demonstrates that neutralizing antibody produced in
response to challenge provides the critical protective component in
pcDNA3JEME-immunized mice. A remarkable difference in postchallenge neutralization titer was observed between mice that survived and mice
that died of lethal challenge. Although some of the surviving mice had
detectable levels of neutralizing antibody before challenge, these
animals were not protected from infection by the challenge virus, since
antibodies to the viral NS1 protein (not encoded by the DNA vaccine)
were detected after challenge. Primary CTLs could also be detected in
pcDNA3JEME-vaccinated mice. However, these CTLs do not appear to be the
critical protective component, since mice which were immunized with a
shorter plasmid and developed CTLs but not neutralizing antibody were
not protected from challenge. On the other hand, partial protection was
observed when antibody collected from pcDNA3JEME-immunized mice 4 days after challenge was administered to naive mice 2 days after JE
challenge. Thus, pcDNA3JEME protects mice from disease but not infection.
It has been reported that mice were partially protected from challenge
by passive transfer of hyperimmune or monoclonal anti-JE virus
antibodies, either before or after lethal challenge (5, 28,
32). We also observed protection in our passive transfer experiments and assume that the protection was incomplete, since insufficient levels of antibody were present or maintained in the
recipient mice. Specifically, the transferred antibody that conferred
partial protection had a titer of 1:320 to 1:1,280 at the time of
injection; thus, the final neutralization titer in the recipient was
expected to be in the range of only 1:32 to 1:128, assuming a serum
volume of 2 ml in mice of this age. Moreover, passively transferred
antibodies may not have a long circulating time in mice (7),
so our partial protection data may be consistent with the circulating
levels of antibody at the time of virus multiplication.
In our mouse model, the challenge virus was injected by an i.p. route,
simulating the primary (peripheral site) and secondary (central nervous
system) growth of virus, which results in encephalitis following
arthropod-mediated transmission. Thus, in both natural infection and
experimental challenge, virus replication at peripheral sites provides
progeny virus capable of stimulating the immune response, as well as
blood-borne virus that can induce the signs and symptoms of the
disease. Based on our estimates of the levels of neutralizing antibody
circulating in mice following challenge (see above), the titers of
antibody present in the sera of pcDNA3JEME-vaccinated mice following
challenge should have been sufficient to provide protection from
encephalitis. On the other hand, unimmunized mice (or control
plasmid-immunized mice) did not produce sufficient neutralization
titers in the face of challenge and were not protected. Thus, a rapid
postchallenge elevation of neutralizing antibody in response to virus
replication, due to the presence of memory B and helper T cells in mice
inoculated with pcDNA3JEME, appears to be the main mechanism of
protection by pcDNA3JEME.
Although two immunizations with 100 µg of pcDNA3JEME provided mice
with postchallenge neutralization titers sufficient for protection,
single immunizations with 100 or 10 µg of pcDNA3JEME elicited
variable outcomes. These data support the importance of a minimum level
of preexisting B cells capable of producing protective levels of
neutralizing antibodies following challenge. Thus, in the
once-immunized mice with insufficient B-cell priming, the rate of virus
replication and spread was able to overwhelm the rising levels of
neutralizing antibodies.
The CTL activity induced by immunization twice with 100 µg of
pcDNA3JEE1/2 did not correlate to the protection under the conditions used in our experiments. As stated above, this result suggests that
postchallenge antibody levels are the primary mediators of protection.
However, passive transfer with virus-specific CTLs obtained from JE
virus-infected mice can protect mice from encephalitis under some
conditions (26), suggesting that CTLs specific for other
epitopes, or larger numbers of CTLs, could be important for protection
from JE. For the dengue viruses, a relatively large number of CTL
epitopes have been identified in NS proteins (2, 18),
although their ability to protect animals from disease has not yet been
determined. Immunization with plasmid DNA encoding portions of the JE
virus polyprotein may prove a particularly effective way to determine a
protective role of individual CTL epitopes encoded by other regions of
the genome.
Passive protection against flavivirus infection in mice has been
reported with IgM class antibodies raised in response to viral
infection (23) and with monoclonal antibodies to NS1
(3). In the present study, since all postchallenge samples
collected from protected mice contained both specific IgM and IgG
antibodies, we cannot state with certainty which isotypes of antibody
were critical for protection. However, sera that had a maximum level of
IgM antibody on day 4 did not show high neutralizing activity (<1:20),
and mice with this serological profile did not survive challenge. A
role for antibody to NS1 in protection cannot be ruled out in our
studies; however, antibodies to NS1 rose similarly in all groups, in
contrast to the enhanced secondary responses to prM/E in the
DNA-vaccinated animals that were protected.
In conclusion, the critical components for protection of mice from JE
appear to be preexisting memory B and T cells which produce sufficient
levels of neutralizing antibodies following virus encounter. The
presence of detectable prechallenge neutralizing antibody was not
always required for protection from death. These conclusions may be
applicable to humans, swine, and horses as well. In the case of human
infection, the existing JE vaccine, which is considered to provide
protection from challenge, does not stimulate 100% seroconversion in
vaccinated populations (4). Moreover, JE is considered to be
endemic in Japan, where most of the population receives this vaccine in
childhood. Although most healthy adults below the age of 60 do not
receive booster immunizations, disease is very rare. One explanation
for this finding is that these individuals are naturally boosted by
periodic bites from JE virus-infected mosquitoes (28). In a
very small study, we found evidence of this type of boosting, due to
the presence of antibodies to NS1 in two of three Japanese who had been
vaccinated and lived in Japan (8). Thus, it seems highly probable that secondary neutralizing antibody responses can protect humans from the disease following the bite by infected mosquitoes. In
general, it is considered that the inactivated vaccine induces neutralizing antibody that protects humans from infection. The protective immunity against infection might be provided when high antibody levels are maintained; however, it is much more likely that
this vaccine induces memory cells that produce a secondary immune
response following infection and that the produced antibodies limit
infection and stop disease. In this sense, the current inactivated JE
vaccines protect by the same mechanism as tick-borne encephalitis vaccines, in that they do not provide protection from infection (17). The continued elucidation of mechanisms for protection against flavivirus disease will be important for development of new
types of vaccines.
| |
ACKNOWLEDGMENTS |
This investigation received financial support from the Vaccine
Research and Development Unit of the WHO Global Programme for Vaccines
and Immunization, from Research on Emerging and Re-emerging Infectious
Diseases, Ministry of Health and Welfare of Japan, and from the Faculty
of Health Science, Kobe University School of Medicine.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan. Phone and Fax: 81-78-796-4594. E-mail:
ekon{at}ams.kobe-u.ac.jp.
Present address: Hyogo Prefectural Institute of Public Health, Kobe
652-0032, Japan.
Present address: Department of Virology 1, National Institute of
Infectious Diseases, Tokyo 162-8640, Japan.
§
Present address: Department of Neurology, Kinki University School
of Medicine, Osaka-sayama 589-8511, Japan.
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Abstract
Introduction
