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J Virol, June 1998, p. 4925-4930, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Induction of Protective Immunity against Japanese
Encephalitis in Mice by Immunization with a Plasmid Encoding Japanese
Encephalitis Virus Premembrane and Envelope Genes
Eiji
Konishi,1,2,*
Masaoki
Yamaoka,1
Khin-Sane-Win,1
Ichiro
Kurane,3 and
Peter W.
Mason4
Department of Medical Zoology, Kobe
University School of Medicine, Kobe 650,1
Department of Health Sciences, Kobe University School of
Medicine, Kobe 654-01,2 and
Department of Microbiology, Kinki University School of
Medicine, Osaka-Sayama 589,3 Japan, and
Plum Island Animal Disease Center, Agricultural Research
Service, U.S. Department of Agriculture, Greenport, New York
119444
Received 5 January 1998/Accepted 24 February 1998
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ABSTRACT |
A DNA vaccine plasmid containing the Japanese encephalitis (JE)
virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME)
was evaluated for immunogenicity and protective efficacy in mice. Two
immunizations of 4-week-old female ICR mice with pcDNA3JEME by
intramuscular or intradermal injections at a dose of 10 or 100 µg per
mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20
(90% plaque reduction), and all immunized mice survived a challenge
with 10,000 50% lethal doses of the P3 strain of JE virus. A single
immunization with 100 µg of pcDNA3JEME did not elicit detectable NEUT
antibodies but induced protective immunity. Spleen cells obtained from
BALB/c mice immunized once with 10 or 100 µg of pcDNA3JEME contained
JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice
maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 µg.
The CTLs induced in BALB/c mice immunized twice with 100 µg of
pcDNA3JEME were CD8 positive and recognized mainly the envelope
protein. These results indicate that pcDNA3JEME has the ability to
induce a protective immune response which includes JE virus-specific antibodies and CTLs.
| |
INTRODUCTION |
One of the recent promising
strategies in protection from viral diseases is the induction of
protective immunity by the expression of subsets of viral genes in the
vaccinated host. This strategy can eliminate immune responses to
unneeded or adventitious antigens present in inactivated virus vaccine
preparations and may provide improved safety relative to live
attenuated virus vaccines. The introduction of subsets of viral genes
into a vaccinee can be accomplished with a recombinant virus
(32) or with naked DNA molecules designed to express the
genes in the cells of the host (22).
We have studied Japanese encephalitis (JE) as a model for
understanding the immunogenicity and protective efficacy
conferred on murine, porcine, and human hosts by different
flavivirus gene products. In these studies, we showed that recombinant
poxviruses carrying the signal sequence for the premembrane (prM), the
prM gene, and the envelope (E) gene express proper forms of the prM and
E proteins in infected cells and that infected cells release these
viral proteins in a particulate form (15, 25). These extracellular particles are morphologically and biochemically similar
to the authentic subviral particles, so-called slowly sedimenting
hemagglutinin, released from JE virus-infected cells (17).
The similarity of these genetically engineered products to natural
virus particles is consistent with our early work showing the
excellent performance of vaccinia virus-based vaccines specific for
these particles in mice (15, 25). Furthermore, a
recombinant poxvirus carrying the same signal sequence-prM-E cassette
but based on a highly attenuated vaccinia virus strain (NYVAC) induced high levels of neutralizing (NEUT) antibodies (16) and
specific cytotoxic T lymphocytes (CTLs) in mice (13) and
protected mice from lethal challenge and swine from viremia
(16). However, this NYVAC-based recombinant poxvirus did not
induce NEUT antibodies to JE virus in vaccinia virus-preimmune
vaccinees in a clinical phase I trial, although it did elicit anti-JE
virus antibodies in vaccinia virus-naive vaccinees (14).
The adverse effect of antivector immunity to the immunogenicity of the
products specified by the vector has been pointed out with several
systems (2, 8, 33) and may cause significant problems for
the viral vector-based strategy, especially in long-lived species, such
as humans. Naked DNA vaccines, which do not suffer from the problem of
antivector immunity, recently have been developed and tested for a
variety of viral pathogens (3, 31, 34-36). Recently, naked
DNA vaccine candidates have been reported for two flavivirus diseases.
Work with St. Louis encephalitis showed that a plasmid carrying the prM
and E genes could induce partial protection in mice, but induction of
NEUT antibodies and CTLs was not demonstrated (28). Another
plasmid containing the prM gene and part of the E gene of dengue type 2 virus induced NEUT antibodies, but protection was not demonstrated
(12). In this report, we studied the immunogenicity and
protective efficacy of plasmid DNA containing the signal sequence-prM-E
cassette of JE virus genes that we had identified to be the most
effective immunogen in poxvirus-based recombinant viral vaccines for
JE.
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MATERIALS AND METHODS |
Construction of plasmids.
The JE virus cDNA containing the
prM signal sequence, the prM gene, and the E gene was amplified by
PCR with DNA template plasmid pARJa (containing Nakayama strain C
protein cDNA sequences fused to plasmids PM-7 and PM-6
[26]; GenBank accession no. M73710). The sense primer
included an EcoRI site, an efficient eukaryotic initiation
site (19), and a start codon, followed by the codons
encoding Glu-Gly-Ser of the prM signal sequence. The antisense primer
corresponded to the C-terminal six codons of the E gene, a termination
codon, and an XhoI site. To facilitate "error-free"
amplification, the selected JE virus coding region was amplified in two
portions, which were combined by use of an artificial EcoRV
site that was added within the coding region (codons 67 and 68 of the E
protein) without changing the encoded amino acid sequence. 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
construct was designated pcDNA3JEME. Proper insertion of the gene
cassette in pcDNA3JEME was confirmed by sequencing with a DNA sequencer
(ABI 373A; Applied Biosystems, Chiba, Japan). pcDNA3JEME DNA was
purified with a Qiagen Plasmid Kit (Funakoshi Co. Ltd., Tokyo, Japan)
following the manufacturer's instructions and was used for
immunization of mice.
Viruses.
The prototype Nakayama strain of JE virus
(23) was used in all in vitro studies, including NEUT tests,
spleen cell stimulation, and cytotoxicity assays. The Nakayama strain,
which exhibits low lethality in many strains of mice, was also used to
"vaccinate" mice. The Beijing P3 strain of JE virus, which is
reproducibly virulent in mice over 6 weeks of age, was used for mouse
challenge studies (25). Recombinant vaccinia viruses used
for infection of target cells in cytotoxicity assays were vP555,
carrying the prM, E, and NS1 genes of the Nakayama strain of JE virus;
vP658, carrying the E and NS1 genes; vP829, carrying the prM and E
genes; and their parent virus, vP410 (15).
Mouse experiments.
Groups of five 4-week-old female ICR mice
were used for evaluating the induction of NEUT antibodies and
protective immunity, and groups of two 6-week-old male BALB/c mice were
used mainly for evaluating the induction of CTLs. Mice were immunized
with pcDNA3JEME at doses of 0.1 to 100 µg, pcDNA3 at a dose of 100 µg, or phosphate-buffered saline (PBS) once or twice at an interval of 2 weeks. The injection route was intramuscular (i.m.) at both thighs
or intradermal (i.d.) at the base of the tail. At 2 or 3 weeks after
immunization, the ICR mice were bled retro-orbitally, and serum samples
were isolated from blood, pooled, and used for evaluation of antibody.
The ICR mice were also challenged by intraperitoneal (i.p.) injection
with 10,000 50% lethal doses (LD50) of the P3 strain of JE
virus and observed for 3 weeks. Postchallenge blood was collected from
mice that survived the challenge. Spleen cell suspensions were prepared
from BALB/c mice as previously described (13) and stimulated
with JE virus for cytotoxicity assays (see below). For examination of
the duration of NEUT antibodies and memory B cells and CTLs, BALB/c
mice that had received one inoculation with pcDNA3JEME at a dose of 100 µg were kept for 1 to 6 months before sample collection.
NEUT tests.
Specific antibodies elicited in immunized mice
were evaluated by NEUT tests as previously described (15).
The NEUT titer was expressed as the serum dilution yielding a 90%
reduction in plaque number.
Cytotoxicity assays.
Stimulation of spleen cells with JE
virus in vitro and cytotoxicity assays were performed as previously
described (13) with some modifications. Spleen cells (4 × 106) were stimulated by incubation with live JE virus
antigen at a final dilution of 1:8 in 2 ml of RPMI 1640 medium
containing 10% fetal bovine serum (RPMI-10% FBS) per well of 24-well
microplates at 37°C for 6 days. The live virus antigen used was
clarified culture fluid harvested from infected C6/36 cell cultures and contained a titer of approximately 2 × 108 PFU/ml in
the undiluted stock, as titrated on Vero cell cultures. The control
antigen used was culture fluid from mock-infected C6/36 cell cultures.
Both live virus and control antigens were used at 1:8 dilutions.
Following the 6-day stimulation step, the cells were washed three times
with RPMI-10% FBS and 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 primary mouse kidney (PMK) cells prepared from kidneys of BALB/c
mice or P815 mastocytoma cells. PMK cells were infected with JE virus
at a high multiplicity of infection (approximately 100 to 200 PFU/cell)
or mock infected 17 to 18 h before the assay, and P815 cells were
infected with vP829 or vP410 at a multiplicity of infection of 10 PFU/ml or mock infected 15 to 20 h before the assay. For target
protein analysis, P815 cells infected with vP555 and vP658 were also
used. 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 with the following formula: 100 × [(experimental release 
minimum release)/(maximum release minimum release)]; the maximum release was obtained by lysing all
the target cells with Renex, and the minimum release was obtained with
target cells incubated alone in RPMI-10% FBS.
Cell depletion assays.
Cell depletion tests were performed
as previously described (13). Briefly, cells stimulated with
JE virus were incubated with antibodies to CD3, CD4, and CD8 at
dilutions of 1:5 to 1:100 at 4°C for 30 min. These cells were then
treated with rabbit complement at a 1:10 dilution at 37°C for 1 h and used in cytotoxicity assays. Cytotoxicity was compared with that
obtained with JE virus-stimulated cells treated only with complement
and with JE virus-stimulated and mock-stimulated cells without
treatment.
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RESULTS |
Proper expression of pcDNA3JEME.
COS7 and Vero cells were
transfected with pcDNA3JEME by use of liposomes (Lipofectin; Life
Technologies Inc., Gaithersburg, Md.) or lipopolyamine
(Transfectam; Biosepra, Villeneuve-ia-Garenne, France), and expression
was determined by indirect fluorescent-antibody staining with a
monoclonal antibody to the JE virus E protein (J3-11B9)
(24). When transfection protocols recommended by
the manufacturer were used, E antigen could be detected with
this antibody in 3 to 5% of either COS7 or Vero cells 1 to 2 days
after transfection, indicating that pcDNA3JEME expressed the E antigen in these eukaryotic cells.
Induction of NEUT antibodies and protective immunity.
ICR mice were inoculated i.m. or i.d. with 10 or 100 µg of
pcDNA3JEME at 4 and 6 weeks of age (Table
1). At 2 weeks after each immunization
(at 6 and 8 weeks of age), sera were collected and checked for
NEUT antibodies. Following the second serum collection, these mice were
challenged with 10,000 LD50 of the P3 strain of JE
virus and observed for 3 weeks; sera were collected at the end of the
3-week observation period from all mice that survived the challenge (at
11 weeks of age). The results in Table 1 show that one immunization
with pcDNA3JEME did not induce detectable levels of NEUT antibodies but
that two immunizations induced NEUT antibodies at titers of 1:10 to
1:20, irrespective of the immunization route and the dose. All mice
given two inoculations survived the challenge but displayed
significantly increased serum NEUT titers (1:160 to 1:640) at 3 weeks
postchallenge. Mice inoculated with PBS did not have detectable levels
of NEUT antibodies and died from the challenge, whereas mice vaccinated
by inoculation with the Nakayama strain of JE virus 4 weeks prior to
challenge had a high NEUT antibody titer and survived the P3 virus
challenge. Since three of five mice died following infection with
5 × 106 PFU of the Nakayama strain of JE virus, this
"vaccinating" dose corresponded to approximately 1 LD50. These results indicate that two immunizations with
pcDNA3JEME induced NEUT antibodies and protective immunity in mice.
Since a significant difference in NEUT antibody titer and survival rate
was not observed between the i.m. and i.d. routes, we used the i.m.
route for subsequent experiments.
Next, the ability of pcDNA3JEME to induce protective immunity in ICR
mice was examined in the one-immunization protocol at 0.1 to 100 µg (Table 2). Immunization was
performed at 4 weeks of age, with bleeding and challenge at 7 weeks, observation for 3 weeks, and postchallenge bleeding when
the mice were 10 weeks old. Nonimmune control groups
included mice inoculated with PBS or pcDNA3 at 100 µg.
All mice immunized with pcDNA3JEME at 0.1 to 10 µg, pcDNA3, and PBS
died from challenge, but partial protection was observed for mice
immunized with pcDNA3JEME at 100 µg. NEUT antibodies were not
observed for prechallenge sera for any of these groups, but
a high NEUT antibody titer was observed for surviving mice immunized
with 100 µg of pcDNA3JEME.
Induction of specific CTLs.
In order to study cellular
responses in mice immunized with pcDNA3JEME, spleen cells were obtained
from BALB/c mice immunized with pcDNA3JEME at 100 µg twice at a
2-week interval, stimulated in vitro with JE virus, and examined
for cytotoxic activity against PMK cells infected with JE virus.
Figure 1 shows the results obtained at an
E/T ratio of 100:1. A high percentage of specific lysis of JE
virus-infected cells (approximately 50%) was obtained with effector
cells stimulated with JE virus but not with unstimulated effector
cells. Cytotoxic activities against mock-infected cells were low. This
result indicates that immunization with pcDNA3JEME induced JE
virus-specific memory CTLs in mice.
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FIG. 1.
Lysis of JE virus-infected PMK cells by
pcDNA3JEME-immune spleen cells stimulated with JE virus. BALB/c mice
were immunized with pcDNA3JEME at 100 µg twice at an interval
of 2 weeks. At 2 weeks after the second immunization, the spleen cells
were harvested and stimulated by incubation with C6/36-grown virus
(JEV) or culture fluid from uninfected C6/36 cells (None) for 6 days.
Cytotoxic activities against JE virus-infected (JEV) or mock-infected
(Mock) PMK cells were measured at an E/T ratio of 100:1 by the standard
chromium release method (see Materials and Methods for details).
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The ability of pcDNA3JEME to induce specific CTLs in BALB/c mice was
examined in the one-immunization protocol at 0.1 to 100
µg (Fig.
2). Specific cytotoxic activities
were observed for mice
immunized with 10 or 100 µg of
pcDNA3JEME. However, responses
in mice immunized with 1 or 0.1 µg of this DNA were not significantly
different from the responses
detected in samples collected from
mice inoculated with 100 µg of
pcDNA3 or PBS.
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FIG. 2.
Lysis of P815 cells infected with a recombinant vaccinia
virus carrying the prM and E genes (vP829) or the parental virus
(vP410) by pcDNA3JEME-immune spleen cells stimulated with JE virus.
BALB/c mice were immunized once with pcDNA3JEME at 0.1 to 100 µg, pcDNA3 at 100 µg, or PBS. At 3 weeks
postimmunization, the spleen cells were harvested and
incubated with C6/36-grown virus for 6 days. Cytotoxic activities were
measured at the indicated E/T ratios by the standard chromium release
method (see Materials and Methods for details).
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Characterization of CTLs.
The phenotype of cells responsible
for cytotoxic activities was determined by use of cell depletion tests
(Fig. 3). Spleen cells obtained from
BALB/c mice immunized twice with 100 µg of pcDNA3JEME and
stimulated in vitro with JE virus were treated with antibodies against
cell surface markers and complement before cytotoxicity assays.
Cytotoxic activities were reduced by treatment with anti-CD3 or
anti-CD8 in the presence of complement, whereas treatment with
complement alone or with anti-CD4 and complement did not reduce the
activities. These results indicate that CD8-positive and CD4-negative T
lymphocytes were responsible for cytotoxic activities.
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FIG. 3.
Phenotypic analysis of CTLs by cell depletion with the
indicated antibodies and complement. BALB/c mice were immunized with
pcDNA3JEME at 100 µg twice at an interval of 2 weeks. Four weeks
later, the spleen cells were harvested and incubated with C6/36-grown
virus for 6 days. After cell depletion (see Materials and Methods for
details), cytotoxic activities were measured at an E/T ratio of 120:1
with 51Cr-labeled P815 cells infected with vP829. JEV, JE
virus; Mock, mock infected.
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We investigated JE virus proteins recognized by CTLs by using
P815 cells infected with recombinant vaccinia viruses expressing
different JE virus antigens (Fig.
4).
Spleen cells obtained from
BALB/c mice immunized twice
with 100 µg of pcDNA3JEME and stimulated
in vitro with JE virus
were used for this experiment. Specific
cytotoxic activities were
observed against target cells infected
with vP555, vP658, and vP829;
all of these recombinant viruses
expressed E protein with or without
prM protein. These results
suggest that the predominant CTLs induced in
pcDNA3JEME-immunized
mice recognized E protein. Since target cells
expressing prM protein
alone were not used in this experiment, the
presence or absence
of a minor population of CTLs which specifically
recognize prM
protein could not be determined.
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FIG. 4.
Target antigen analysis of CTLs. BALB/c mice were
immunized with pcDNA3JEME at 100 µg twice at an interval of 2 weeks.
At 3 weeks later, the spleen cells were harvested and incubated with
C6/36-grown virus for 6 days. Cytotoxic activities were measured at the
indicated E/T ratios with 51Cr-labeled P815 cells infected
with a recombinant vaccinia virus encoding no antigens (parental virus
vP410), prM, E, and NS1 (vP555), E and NS1 (vP658), or prM and E
(vP829) or mock infected (Mock). JEV, JE virus.
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Duration of immunity induced by pcDNA3JEME.
The levels of NEUT
antibodies and protective immunity were examined with groups of two
male BALB/c mice at 1 to 6 months after one immunization with
pcDNA3JEME at 100 µg. Since undetectable levels of NEUT antibodies
were found in ICR mice 3 weeks after one immunization (at 7 weeks of
age; Table 2), the BALB/c mice in this experiment testing duration of
immunity were challenged at different intervals following immunization,
and the induction of NEUT antibodies at 4, 8, and 21 days after
challenge was used as an indicator of the presence of
vaccination-induced memory B cells (Table
3). As expected, prior to challenge (day
2 in Table 3), only low or undetectable NEUT antibody titers were observed for each pair of mice immunized with pcDNA3JEME, and no NEUT
antibodies were detectable in mice inoculated with PBS. Following
challenge, the NEUT antibody titers in mice immunized with
pcDNA3JEME were elevated to 1:160 to 1:640 by day 4 and to 1:320 to
1:1,280 by day 8, and these levels were maintained or further increased
until day 21. On the other hand, the titers of NEUT antibodies in
unimmunized mice remained low (undetectable or 1:10) until days 4 and
8. All unimmunized mice died before day 10, and all immunized mice
survived throughout the observation period (21 days). Although only two
mice were used per group in this experiment, the results indicate that
mice immunized with pcDNA3JEME maintained sufficient memory B cells to
supply high titers of NEUT antibodies if challenged within 6 months of
immunization.
The levels of memory CTLs were examined with two male BALB/c
mice at 6 months after one immunization with pcDNA3JEME at 100
µg.
The percentages of specific lysis obtained against vP829-
and
vP410-infected targets were 39.3 and 13.6% at an E/T ratio
of 400:1
and 23.9 and 5.4% at an E/T ratio of 200:1, respectively.
This result
indicates that mice immunized with pcDNA3JEME maintained
detectable
levels of memory CTLs for at least 6 months.
| |
DISCUSSION |
This paper demonstrates that the JE virus prM and E genes
introduced into mice in the form of plasmid DNA induced NEUT
antibodies, CTLs, and protective immunity. In flavivirus
infections, the prM, E, and NS1 proteins have been considered to induce
protective immunity, since protection of mice from lethal challenge has
been shown by passive transfer of monoclonal antibodies against the prM
(10), E (11, 24) and NS1 (7) proteins.
Recently, several epitopes on other viral proteins important for the
induction of cellular immunity, including CTLs, were analyzed (5,
20, 21), but no epitopes were demonstrated to be responsible for protection. Although passive transfer of JE virus-specific CTLs protected mice from lethal challenge (27), the epitopes
recognized by these CTLs were not characterized.
Our previous studies with poxvirus-based recombinant JE viruses
demonstrated the proper synthesis of intracellular and extracellular forms of prM and E proteins in cells infected with recombinants encoding the signal sequence of prM, prM, and E, independent of the
vector virus used: vaccinia virus (15, 16), canary poxvirus (18), and Sindbis virus (30). Furthermore, a
similar cassette can be used to synthesize extracellular particles
containing the structural proteins of other flaviviruses, including
yellow fever virus (29), dengue type 1 virus (4),
and tick-borne encephalitis virus (1). In the present study,
we showed that a plasmid carrying these JE virus genes could be used to
produce E protein in COS7 and Vero cells. Since the in vitro
transfection efficiency of pcDNA3JEME was low in these cells, we did
not attempt to identify extracellular forms of E in transfected cell
cultures. However, the proper synthesis of E in the transfected cells
was supported by the induction of specific antibodies, CTLs, and
protective immunity in mice inoculated with pcDNA3JEME.
Several devices to increase the level of expression of JE
virus-proteins were incorporated into pcDNA3JEME. We chose a
vector with a strong eukaryotic promoter derived from human
cytomegalovirus and a well-characterized polyadenylation signal derived
from bovine growth hormone. We inserted a strong eukaryotic initiation
site containing an ACC sequence which precedes the AUG start codon and
which has been reported to be an optimal sequence for initiation by
eukaryotic ribosomes (19). We also altered the prM signal sequence to enhance expression. Specifically, we added 5 amino acids to
the 15-amino-acid sequence that we used in earlier poxvirus recombinants (15, 25), based on results which were obtained with recombinant vaccinia viruses encoding similar cassettes for other
flavivirus genomes and which showed that longer prM signal sequences
resulted in higher levels of synthesis of extracellular particles (data
not shown). Furthermore, we did not include the gene for NS1 in
pcDNA3JEME, since the production of extracellular particles (which we
believe are the critical immunogens) from cells infected with vP829
carrying the prM and E genes was eight times higher than the production
with vP555 carrying the prM, E, and NS1 genes and since vP829 induced
higher levels of protective immunity than vP555 in mice
(15).
Induction of CTLs is one of the prominent features of DNA immunization.
Cumulative experimental data have established a theory that peptides
expressed by foreign genes introduced into cells are bound to
major histocompatibility complex class I molecules and are
recognized by CD8-positive T lymphocytes, including CTLs (6). In the present study, memory CTLs were
demonstrated in pcDNA3JEME-immunized BALB/c mice. CTLs induced by
pcDNA3JEME immunization recognized mainly E protein, consistent with
our previous data indicating that recombinant poxviruses carrying
prM, E, and NS1 proteins induced CTLs that recognized mainly E protein
(13). Interestingly, mice immunized once with 100 µg of
pcDNA3JEME, which did not induce high levels of NEUT antibodies,
were protected from lethal challenge. Immunization with the same dose
of pcDNA3JEME induced CTLs in BALB/c mice, making it tempting to
speculate that the protection observed in animals given a single dose
of 100 µg was due to CTL responses. However, it is possible that low levels of antibodies (below the detection limit in our assay) present
prior to challenge or antibodies produced by memory B cells and helper
T cells that were rapidly activated following i.p. exposure to the
challenge virus may have been responsible for protection. Current
studies are aimed at determining the components that confer protection
in our murine challenge system.
Consistent with the current view on protection from cytopathic viruses
(9), studies of JE virus suggest that preexisting antibodies
provide the critical and predictive factor in protection. In our
previous experiments, recombinant vaccinia viruses that express the E
protein synthesized in a misfolded form in infected cells failed to
induce NEUT antibodies and provided little protection from challenge
(25). In the present study, using DNA vaccines, we
discovered an immunization strategy in which animals with low or
undetectable levels of NEUT antibodies were protected from challenge.
Following challenge, the sera from these animals contained high levels
of NEUT antibodies, indicating a significant secondary immune response
due to the challenge virus (probably due to replication at peripheral
sites). We previously reported the replication of challenge virus in
mice which had high prechallenge NEUT titers against JE virus
(15), indicating that sterile immunity may be difficult to
achieve in our challenge system. Nevertheless, the data presented in
this paper suggest that protection induced by delivery of JE virus gene
subsets by recombinant viruses or as naked DNA may result from a
different mechanism. Thus, in addition to its usefulness as a
vaccine candidate, naked DNA immunization could be useful for
elucidating the mechanisms of protection against flavivirus diseases.
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ACKNOWLEDGMENTS |
This investigation received financial support from the Vaccine
Research and Development Unit of the WHO Global Programme for Vaccines
and Immunization and from Research on Emerging and Re-emerging Infectious Diseases, the Ministry of Health and Welfare of Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-01, Japan. Phone: 81-78-796-4594. Fax:
81-78-796-4594. E-mail: ekon{at}ams.kobe-u.ac.jp.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
