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Journal of Virology, November 1998, p. 8472-8476, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Canine Distemper Virus DNA Vaccination Induces
Humoral and Cellular Immunity and Protects against a Lethal
Intracerebral Challenge
Nathalie
Sixt,
Alicia
Cardoso,
Agnès
Vallier,
Joël
Fayolle,
Robin
Buckland, and
T. Fabian
Wild*
Unité INSERM 404, Immunity and
Vaccination, Bâtiment Ex-Institut Pasteur de Lyon, 69365 Lyon
Cedex 07, France
Received 11 May 1998/Accepted 6 August 1998
 |
ABSTRACT |
We have studied the immune responses to the two glycoproteins of
the Morbillivirus canine distemper virus (CDV) after DNA vaccination of BALB/c mice. The plasmids coding for both CDV
hemagglutinin (H) and fusion protein (F) induce high levels of
antibodies which persist for more than 6 months. Intramuscular
inoculation of the CDV DNA induces a predominantly immunoglobulin G2a
(IgG2a) response (Th1 response), whereas gene gun immunization with CDV
H evokes exclusively an IgG1 response (Th2 response). In contrast, the CDV F gene elicited a mixed, IgG1 and IgG2a response. Mice vaccinated (by gene gun) with either the CDV H or F DNA showed a class
I-restricted cytotoxic lymphocyte response. Immunized mice challenged
intracerebrally with a lethal dose of a neurovirulent strain of CDV
were protected. However, approximately 30% of the mice vaccinated with
the CDV F DNA became obese in the first 2 months following the
challenge. This was not correlated with the serum antibody levels.
| |
INTRODUCTION |
Inoculation of plasmid DNA into
muscle and the subsequent expression of the encoded protein have opened
up new approaches in vaccination and gene therapy (for an extensive
review see reference 25). Although initial studies
used intramuscular (i.m.) inoculation to deliver the DNA, other routes
have been shown to be equally or more efficient in inducing immune
responses, which may be related to the types of antigen-presenting
cells (APCs) which are involved (9). Recent observations
suggest that after i.m. inoculation, muscle cells probably act as a
reservoir for the foreign antigen, while the bone marrow cells seem to
act as the APCs (8, 12, 26, 27). For DNA delivery to the
skin, the APCs have not yet been identified but could well include
cells of dendritic origin (21). Although both intradermal
and i.m. DNA inoculations induce a strong Th1 response, inoculation of
DNA precipitated onto gold beads and delivered by means of a gene gun
favors a Th2 response (7). Whether this is due to the
targeting of different APCs has not been determined.
We have been studying the possibility of using DNA vaccination to
protect against canine distemper virus (CDV). CDV is a member of the
genus Morbillivirus, in the Paramyxoviridae
family, and although this virus primarily infects dogs, the disease has
also been described in several animal species both in nature and in captivity (10, 18, 22). The currently available live
attenuated vaccine efficiently protects dogs once maternal antibodies
have disappeared, but it is not sufficiently attenuated for certain other animal species in which a fatal infection may ensue
(6). This has led to a problem in protecting members of rare
animal species living in captivity.
In the present study, we have expressed the two CDV glycoproteins, the
attachment protein (hemagglutinin [H]) and the fusion protein (F),
from plasmids driven by a cytomegalovirus (CMV) promoter. We show that
i.m. and intradermal inoculation of the CDV H-encoding plasmid induces
a Th1 response, whereas gene gun inoculation of the same plasmid
induces a Th2-type response. In contrast, the CDV F gene administered
with the gene gun elicited a mixed Th response. Mice immunized with
either of the plasmids were protected against a lethal intracerebral
(i.c.) infection.
| |
MATERIALS AND METHODS |
Plasmid construction.
cDNAs encoding CDV H and CDV F were
subcloned into the BglII site of the pV1J plasmid
(17), which is driven by the CMV promoter. To
facilitate making these constructions, site-directed mutagenesis (15) was used to introduce BamHI sites at the 5'
and 3' ends of the CDV F cDNA. Furthermore, site-directed mutagenesis
was used to modify the context of the ATG in the CDV F cDNA in order to
optimize the expression of the encoded proteins (14). In vitro expression of pV1J-CDV-H and pV1J-CDV-F was tested by
transient transfection of murine Ltk
cells. Purified plasmid DNAs
were transfected into Ltk cells by Lipofectamine (Gibco BRL,
Cergy Pontoise, France). At 48 h posttransfection, cells were
examined for the production of proteins by immunofluorescence.
Gold bead-DNA preparations.
Approximately 30 mg of
1.6-µm-diameter gold powder (Bio-Rad, Ivry sur Seine, France) was
mixed with 100 µl of 0.1 M spermidine (Sigma). After sonication in a
water bath sonicator, plasmid DNAs were added at 0.1 to 2 µg/mg of
gold powder. Two hundred microliters of 2.5 M CaCl2 was
then added to the mixture with gentle spinning. Pellets were washed
three times and suspended in cold 100% ethanol. Tubes with gold
bead-DNA preparations were stored at 4°C under dry conditions.
DNA immunizations.
Female BALB/c
(H-2d) mice were purchased from IFFA-CREDO
(Domaine des Oncins, France) and immunized at 6 to 8 weeks of age. For
i.m. inoculations, DNA (50 µg) in 50 µl of phosphate-buffered saline (PBS) was inoculated into the quadriceps muscles of both hind
legs. For intradermal inoculations, DNA (100 µg in 50 µl of PBS)
was administered either with a multipuncture ring (similar to those
used for tuberculin tests) in the abdominal skin or by tail
scarification or subcutaneous injection in the abdomen. For gene gun
inoculations, plasmid DNAs were injected into the shaved abdominal
epidermis of the mice with a Helios gene gun system (Bio-Rad) at a
helium pressure setting of 300 lb/in2. Two gene gun
inoculations at different sites were given for each dose. Each
inoculation delivered 0.5 mg of gold bead-DNA preparation.
Viruses and cells.
The Onderstepoort CDV strain was used for
the neutralization assays, and the mouse neuroadapted CDV strain
(2) was used for challenge studies. Stocks of the latter
were prepared by passage in suckling-mouse brains. Mouse brain
homogenates from infected suckling mice were used as a source of virus
and were stored at 80°C. Cytotoxic T-lymphocyte (CTL) assays were
performed in P815 (H-2d) cells (which express
major histocompatibility complex class I but not class II molecules)
and in CDV-infected P815 cells. These cells were also used for
enzyme-linked immunosorbent assays (ELISAs).
Neutralization assay.
Serial twofold dilutions of mouse sera
were prepared in a sterile 96-well flat-bottomed tissue culture plate.
Dilutions were made in Dulbecco's minimal essential medium (DMEM)
supplemented with 10% fetal calf serum (FCS) and gentamicin (50 µg/ml). Fifty microliters of CDV (25 PFU per well) was added to each
well (excluding cell controls), and plates were incubated for 90 min at
37°C. One hundred microliters of a suspension of Vero cells (2 × 105/ml) was added to each well. Plates were incubated
for 4 to 6 days at 37°C.
Antibody assays.
Titers of antibodies against the CDV H and
F proteins were determined by ELISAs. CDV-infected P815 cells were
distributed in 96-well microtiter plates (1.5 × 105
cells per well). Diluted antisera were incubated with the intact cells
for 90 min at 37°C. The specific antibody was developed with
biotinylated anti-mouse immunoglobulin G1 (IgG1) or IgG2a and the
streptavidin-phosphatase alkaline system (Sigma). Results are expressed
with reference to control anti-H or anti-F monoclonal antibodies, which
were used to standardize the assays for IgG1 and IgG2a. Titers were
calculated with the SOFTmax program (Molecular Device Corporation,
Menlo Park, Calif.). Data are given in nanograms of specific antibody
per milliliter of serum sample.
CTL assays.
Spleens were removed from immunized animals and,
after elimination of erythrocytes by perfusion with DMEM, cocultivated
with CDV-infected P815 (H-2d) cells which had
been incubated with mitomicin C (25 µg/ml) for 30 min at 37°C. The
ratio of spleen cells to stimulator cells was 30:1. The cocultures were
distributed in 24-well plates and incubated in DMEM-10% FCS
containing 5 × 10
5 M 2-mercaptoethanol. On day 5 half the medium was changed, and the cytotoxic activity was tested on
day 7.
For the CTL assays P815 cells and CDV-infected P815 cells were
radiolabelled with 50 µCi of 51Cr for 90 min at 37°C,
washed twice in DMEM-1% FCS, incubated for 1 h in DMEM-10%
FCS, and washed once before use in a 4-h 51Cr release
assay. The percent cell lysis was calculated as follows: {[experimental counts per minute (cpm) spontaneous cpm]/[total cpm spontaneous release]} × 100. Results are expressed as
the percent lysis. Spontaneous release and total release were
determined for target cells incubated with medium alone and after the
addition of 100 µl of 1 M HCl, respectively.
Virus challenge.
Immunized mice were challenged by i.c.
inoculation of a neuroadapted CDV strain. Briefly, 3 weeks after the
first immunization, half of the mice in each group received boosters.
Two weeks later, mice were inoculated i.c. with 50 µl of the
neuroadapted strain (5 × 104 PFU/ml). Control animals
consisted of either uninjected mice or mice immunized with blank
vector. Survival was monitored daily for 30 days.
| |
RESULTS |
Humoral and cellular response to pV1J-CDV-H.
The plasmid
PV1J-CDV-H when transfected into either L or HeLa cells elicited a high
level of expression of CDV H at the surface of the cells, as
measured by immunofluorescence (data not shown).
In order to examine the efficiency of this plasmid as an
immunogen in vivo, BALB/c mice were immunized by different routes
of
inoculation. A single i.m. inoculation (100 µg/mouse) induced
high
levels of antibody, mainly of the IgG2a isotype, which increased
to
maximum values by 3 months (Table
1).
Application of the DNA
by tail scarification (100 µg/mouse) or
multipuncture ring induced
lower antibody levels (Table
1).
Whereas scarification induced
mainly IgG2a antibodies, the
multipuncture ring system induced
predominantly IgG1.
To study the efficiency of the gene gun, mice were inoculated with 2, 0.5, or 0.1 µg of pV1J-CDV-H, and groups of mice received
boosters
with the same quantity of plasmid DNA either at 3 weeks
or at 24 weeks. The antibody response after a single inoculation
increased
during the first 6 weeks and then remained stable over
the following 5 months (Table
1). The level of the response was
related to the amount
of DNA inoculated. Booster immunizations
given at 3 weeks or 6 months
increased the antibody titers at
least twofold. After this method
of vaccination only IgG1 antibodies
were detected, IgG2a antibodies
being present at concentrations
of less than 0.1 µg/ml.
When the sera were examined for virus neutralization activity, only
approximately half of the mice immunized with gene gun
and 40% of the
mice immunized i.m. had significant neutralizing
antibody titers (Table
2).
To study the potential of the pV1J-CDV-H to induce CTLs, BALB/c mice
were immunized by the gene gun method and 3 weeks later
their
spleens were removed and the CTL response was examined.
A good class
I-restricted CTL response was observed (Fig.
1A).
Furthermore, no NK activity could be
detected in these effectors
since they did not lyse NK-sensitive YAC-1
targets (data not shown).
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FIG. 1.
Anti-CDV H (A) and F (B) CTL responses 30 days after a
single immunization with pV1J-CDV-H or pV1J-CDV-F (2 µg) by the gene
gun method, respectively. After in vitro stimulation with
CDV-infected p815 cells, lysis was measured for CDV-infected P815 cells
(open symbols) and P815 cells (circles), which were used as a negative
control. The results are the percents lysis for pairs of animals at
graded effector-to-target (E/T) ratios.
|
|
Humoral and cellular response to pV1J-CDV-F.
Transfection of L
or HeLa cells with pV1J-CDV-F yielded a high level of expression of the
CDV F protein at the surface of the cells as measured by
immunofluorescence (results not shown). To investigate the immunizing
potential of this plasmid, BALB/c mice were immunized i.m. with
pV1J-CDV-F at 100 µg/mouse and the antibody responses were measured
after 3 and 7 weeks. Immunization with 20 µg also elicited an
antibody response (data not shown). Antibody concentrations of 1 to 2 µg/ml were induced with both concentrations of DNA by 3 weeks, decreasing slightly by 7 weeks (Table
3). The IgG2a antibody was the
predominant isotype.
Immunization with the gene gun (2, 0.5, or 0.1 µg/mouse) induced
high levels of anti-F antibodies, with increases during a
6-week period
(Table
3). The response was greater with higher
DNA
concentrations. A booster inoculation at 3 or 12 weeks increased
the antibody titers two- to fourfold. The antibody isotype
profiles
were mixed, with both IgG1 and IgG2a being induced.
The different
protocols (DNA concentration and booster inoculation)
did not
alter radically the antibody isotype profile.
Although CTL responses for CDV F have not yet been demonstrated, we
examined the possibility that DNA vaccination with pV1J-CDV-F
may
induce a CTL response. BALB/c mice were inoculated by the
gene
gun method, and the CTL response was examined 3 weeks later.
A class I-restricted CTL response of similar magnitude to that
induced
by the DNA for the H protein was observed (Fig.
1B). Furthermore,
no NK activity could be detected in these effectors, since they
did not lyse NK-sensitive YAC-1 targets (data not shown).
Survival study following i.c. viral challenge.
In order to
evaluate the level of protection induced by the CDV H and F DNAs, mice
were immunized by the gene gun method with 2, 0.5, or 0.1 µg of
pV1J-CDV-H or pV1J-CDV-F. Control mice were either not injected or
inoculated with blank vector. Two weeks after the last immunization,
the mice were challenged i.c. with a lethal dose of a neuroadapted
strain of CDV (Fig. 2). In the pV1J-CDV-H-immunized mice, the protection was almost complete, with
only one of eight mice receiving the lowest DNA concentration dying
(Fig. 2A). The pV1J-CDV-F gave 70 to 90% protection (Fig. 2B).
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FIG. 2.
Survival of mice immunized by the gene gun method
with pV1J-CDV-H (A) or pV1J-CDV-F (B) after lethal i.c. challenge. Mice
were immunized once or twice with pV1J-CDV-H or pV1J-CDV-F at the
indicated doses. Controls either were not injected (closed triangles)
or were inoculated with blank vector (V1J) (closed circles). All
animals were challenged i.c. 2 weeks after the final immunization, and
survival was assessed daily for 30 days.
|
|
We observed previously that with mice inoculated with sublethal doses
of the neuroadapted strain of CDV, 20 to 40% of the
animals became
obese (
2). We therefore kept the vaccinated-challenged
mice under observation for a further 3 months to see if they
developed
such a syndrome. Of the 21 pV1J-CDV-H-immunized
animals, only
one mouse became obese (5%). In contrast, 5 of 16 surviving mice
immunized with pV1J-CDV-F did so (31%) (Table
4). This phenomenon
was not related
either to the concentration of DNA used for vaccination
or to the serum
antibody titers attained (Table
4).
| |
DISCUSSION |
CDV has a wide host range in animals that has recently been shown
to extend to certain members of the Felidae family (lions and tigers) both in the wild and in captivity (18,
22). The currently available CDV vaccines are of the live
attenuated type and are based on passage in either canine or
avian cells. The latter vaccine is more attenuated in dogs, but its
efficiency is lower than the canine-cell-passaged vaccine. Although
both these vaccines have an attenuated phenotype in dogs, they may induce a more virulent infection in other species (3). Thus, a vaccine which does not consist of infectious virus may have certain
advantages in terms of such a risk. However, the mechanisms of
protection that such vaccines induce remain to be investigated.
In the present study, we have examined the possibility of protecting
against CDV infection by DNA vaccination. Our studies on the closely
related measles virus have shown that both humoral and cell-mediated
immunity are important in protection. Antibodies to either of the two
viral glycoproteins neutralize infection in vitro and passively protect
mice in vivo (28). Furthermore, immunization of mice with a
single class I-restricted CTL epitope protects them from a lethal i.c.
dose of CDV (1). In contrast, in a monkey model the CTL
response could not be shown to reduce the virus load.
In the present study, we have examined the effects of the route of DNA
inoculation on the quantity and quality of the immune response against
CDV. Both i.m. and gene gun immunizations induced high levels of
antibodies. In agreement with previous observations obtained with
measles virus glycoproteins (4, 5) and those made by others
with different viral systems, the gene gun induced an IgG1 (Th2)
response, whereas inoculation by the other routes induced an IgG2a
(Th1) response (20, 26). These results do not appear to be
related to the site of inoculation, as CDV H DNA given by the gene gun
directly into the muscle still induced IgG1 antibodies
(24a). Similar results have been reported with influenza
virus H DNA (7). Thus, it was surprising to find that
the CDV F DNA inoculated with the gene gun induced a mixed IgG1
and IgG2a response. This is in contrast to our studies with measles
virus F, which despite having 70% sequence homology with CDV F induces
only IgG1 antibodies. Recent studies have reported that certain DNA
sequences can induce different cytokines and so influence the
subsequent immune response (13, 23, 24). It is possible that
such sequences favoring Th1 cytokines may be present in the CDV F
sequence, and it should therefore be possible to identify them by using
measles virus-CDV-F DNA chimeras.
Although mice immunized with either CDV H or F were protected
from a lethal i.c. challenge, of the CDV F-immunized animals, more than
30% became obese within the 3 months following the challenge. Our
group and others have previously described this phenomenon in
mice infected with sublethal doses of CDV (2, 16), and it seems to result in loss of critical populations of
hypothalamic neurons (19). In our study, an obesity syndrome
was observed in animals challenged with CDV which had been vaccinated
with different quantities of DNA, and this did not correlate with serum antibody levels. The major difference between the two groups of animals was the presence of virus-neutralizing antibodies, detectable in vitro only, for the CDV H-immunized mice. These results are compatible with the previous report by Hirayama et al. (11), but it is not clear if neutralizing antibodies are the main marker for
protection against subsequent virus-induced pathologies or whether an
alternative type of immune response is implicated in protection.
There are numerous examples in which antibodies do not neutralize virus
in vitro but protect against it in vivo and in which they neutralize it
in vitro but do not protect against it in vivo. In the CDV model, it is
necessary to study the contributions of the different types of
immune responses.
| |
ACKNOWLEDGMENTS |
These studies were supported by a grant from the French
Rhône-Alpes Région (Emergence Programme).
We thank C. Orvell for supplying CDV monoclonal antibodies for
calibrating our ELISAs, F. Henry for animal care, and B. Maret for
editorial assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
INSERM 404, Immunity and Vaccination, Bâtiment Ex-Institut
Pasteur de Lyon, Avenue Tony Garnier, 69365 Lyon Cedex 07, France.
Phone: 33 4 72 72 25 53. Fax: 33 4 72 72 25 67. E-mail:
wild{at}lyon151.inserm.fr.
| |
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Abstract
Introduction
