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Journal of Virology, May 2000, p. 4908-4911, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Maternally Transferred Antibodies from
DNA-Immunized Avians Protect Offspring against Hepadnavirus
Infection
Christine
Rollier,
Celine
Charollois,
Catherine
Jamard,
Christian
Trepo, and
Lucyna
Cova*
Unité de Recherche sur les Virus des
Hépatites et les Pathologies Associées, Institut
National de la Santé et de la Recherche Médicale,
Unité 271, Lyon, France
Received 28 December 1999/Accepted 25 February 2000
 |
ABSTRACT |
The outcome and protective efficacy of maternal antibodies elicited
by DNA immunization to the large (L) hepadnavirus envelope protein were
studied using the duck hepatitis B virus (DHBV) model. Following
genetic immunization of breeding ducks with a DHBV L protein
gene-bearing plasmid, specific and highly neutralizing antibodies were
transferred from the sera of immunized ducks, via the egg yolk, to the
progeny of vaccinees. Interestingly, large amounts (60 to 100 mg/egg)
of high-titer and L protein-specific yolk immunoglobulins
(immunoglobulin Y) accumulated in the egg yolk. These results suggest
that eggs from genetically immunized avians may represent a potent
source of DNA-designed antibodies specific to viral antigen.
Importantly, these antibodies are vertically transmitted and protect
offspring against high-titer DHBV challenge.
| |
TEXT |
Genetic vaccination is a promising
novel approach which has been shown to elicit specific immunity against
different pathogens, including hepatitis B virus (HBV) (reviewed in
reference 8). However, it is not known whether
maternal antibodies elicited by DNA immunization against hepadnavirus
envelope proteins may protect progeny against viral infection.
Moreover, the future of antibodies elicited in DNA-immunized avians and
the protection of their offspring have not been investigated. Following
protein vaccination of breeding ducks or chickens, only one class of
maternal immunoglobulin, immunoglobulin Y (IgY), which is considered to be equivalent to mammalian IgG, is transferred from the blood circulation via the egg yolk to the offspring (13). In this regard, there is a growing interest in the avian egg as a potent supplier of antibodies, since following immunization with a given antigen, large amounts of antigen-specific IgY accumulate in the egg
yolk, from which it can be easily isolated in purified form.
In this study we have chosen the duck HBV (DHBV) model to study the
outcome of maternal antibodies elicited by DNA immunization against
large (L) hepadnavirus envelope protein. The major DHBV neutralization
epitopes, which are also involved in host cell interaction, map within
the pre-S region of the 36-kDa L envelope protein (5, 17).
We and others have previously reported that antibodies elicited by
genetic immunization with a plasmid bearing genes expressing the DHBV
envelope proteins neutralize DHBV infectivity when the antibodies are
preincubated with virus before infection of primary duck hepatocytes
(PDHs) or neonatal ducklings (16, 18). We investigated here
the transfer of maternal anti-DHBV envelope antibodies from
DNA-immunized ducks to hatchlings via the egg and their ability to
confer protection to progeny of vaccinees.
Large amounts of specific and neutralizing antibodies can be
purified from egg yolk following DNA immunization of ducks against DHBV
L envelope protein.
First, we investigated whether antibodies
elicited by DNA immunization against DHBV L protein are transmitted to
the egg yolk. Three laying Pekin ducks (Anas domesticus)
were immunized intramuscularly with 100 µg of the recombinant
pCI-preS/S plasmid, which expresses the DHBV L protein, or the empty
pCI plasmid in saline buffer (NaCl, 0.9%) at weeks 4, 7, 10, as
described previously (16), and boosted under the same
conditions with 200 µg of plasmid at week 28. The kinetics of the
anti-preS antibody response were tested by a previously described
enzyme-linked immunosorbent assay (ELISA) (5). The results
showed that DNA immunization of the ducks induced high titers of
antibody that reached a plateau after the first boost (Fig.
1A), but anti-preS antibodies were not
detected in the control ducks immunized with the empty pCI vector (data not shown). Eggs were collected weekly, during six consecutive weeks,
from the laying ducks starting after the last DNA boost. Total
immunoglobulins from egg yolk sac were extracted and purified by using
the EGGstract IgY Purification System (Promega, Charbonnieres, France).
As shown in Fig. 1B, no anti-preS antibodies were detected in eggs laid
by pCI-immunized ducks. In contrast, the yolk antibodies purified from
eggs laid by pCI-preS/S-immunized ducks had high ELISA titers of
anti-preS antibody which paralleled those of the sera of the immunized
ducks and no decrease in titer was seen during six consecutive weeks of
follow-up without additional DNA boosts (Fig. 1B). Importantly, large
amounts, i.e., 60 to 100 mg of purified IgY, were obtained from each
egg yolk, and there was little variation in egg-to-egg anti-preS
antibody titers at each time point.
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FIG. 1.
Follow-up of humoral anti pre-S antibody responses in
sera of DNA-immunized laying ducks and in their eggs. (A) Individual
kinetics of anti-preS antibody titers of three immunized female ducks
following immunization with the pCI-preS/S plasmid. Arrows indicate DNA
injections at weeks 4, 7, 10, and 28. (B) --X--, mean anti-preS
antibody titers in the sera of these pCI-preS/S-immunized ducks after
the last DNA boost (arrow);   , mean anti-preS yolk IgY titers
from two to four eggs laid by these ducks at each time point;  ,
mean anti-preS IgY titers from eggs laid by control pCI-immunized
ducks. Vertical bars represent standard deviations. Detection of
endpoint anti-preS antibody titers, expressed in log 10 units, was
performed by direct ELISA.
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The specificities of egg yolk antibodies obtained from the
DNA-immunized ducks were tested by immunoblot analysis using
concentrated
virions and mock sera from DHBV-infected and noninfected
duck
sera, respectively, followed by the detection of bound antibody
with anti-rabbit or anti-duck immunoglobulin peroxydase-labeled
antibody and a chemiluminescence detection kit (Amersham, Courtaboeuf,
France), as described previously (
3). The results showed
that
IgY from eggs laid by pCI-preS/S-immunized ducks recognized the
36-kDa DHBV L envelope protein in concentrated, serum-derived
viral
particles (Fig.
2C). The immunoblotting
pattern was comparable
to the one obtained with the sera from breeding
ducks immunized
with this plasmid (Fig.
2B) or with the control serum
from a rabbit
immunized with the recombinant DHB preS envelope protein
(Fig.
2A). The IgY from eggs laid by pCI-immunized ducks were not
reactive
in this test (Fig.
2D).
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FIG. 2.
Specificity of purified IgY. Proteins from DHBV-infected
(lanes marked "Virions" and noninfected (lanes marked "Mock")
duck sera were revealed in an immunoblotting assay with polyclonal
anti-DHB preS rabbit antiserum (A), serum from pCI-preS/S-immunized
ducks (B), IgY from an egg laid by a pCI-preS/S-immunized duck (C), and
IgY from an egg laid by a pCI-immunized duck (D) (control). The p36
DHBV L protein is indicated.
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It was important to assess whether such egg yolk antibodies from
DNA-vaccinated ducks are neutralizing. To this end, the capacity
of
these antibodies to neutralize DHBV infection was tested in
vitro with
PDHs obtained by in situ collagenase perfusion (
3).
The PDHs
were infected with a DHBV inoculum (2 × 10
8 virus
genome equivalents [vge]/well) which had been preincubated
overnight
at room temperature with either phosphate-buffered saline,
duck serum,
or purified egg yolk IgY. As illustrated in Fig.
3,
a total virus release, reflected by
the area under the curve,
was not affected by preincubation of the
inoculum with either
control duck serum or control yolk IgY from eggs
laid by empty
pCI vector-immunized ducks (Fig.
3). By contrast, serum
from ducks
immunized with the pCI-preS/S plasmid and IgY from eggs laid
by
these ducks were highly neutralizing, since decreases by 96 and
99%, respectively, of released virions were observed in PDHs infected
with DHBV preincubated with these antibodies (Fig.
3). Taken together,
these results indicate that genetic immunization of ducks with
the
plasmid bearing the gene encoding hepadnavirus L envelope
protein
elicits large amounts of antigen-specific and highly neutralizing
egg
yolk antibodies.
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FIG. 3.
In vitro neutralization of DHBV infectivity with
immunized-duck sera and egg yolk IgY. PDHs were infected with DHBV
inoculum preincubated with phosphate-buffered saline
( ), sera from pCI-immunized
ducks
( ), egg
yolk IgY from pCI-immunized ducks
( ), sera
from pCI-preS/S-immunized ducks
(------), and IgY from
pCI-preS/S-immunized ducks
(---×---).
The release of viral particles was quantified in cell medium in virus
genome equivalents by dot blot hybridization. Means of duplicate
determinations are presented.
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Offspring of DNA-vaccinated ducks are protected against high-titer
DHBV challenge.
To verify that maternal antibodies transmitted to
the eggs reached the progeny circulation, fecund eggs from DNA
vaccinees were placed in an SMA 120 incubator (SOCMA, Beaumont, France) and incubated at 37.5°C and 55% humidity during 28 days until hatching. Our results show that none of the ducklings from
pCI-immunized ducks had detectable anti-preS antibodies at hatch as
assessed by ELISA but that all ducklings from pCI-preS/S-immunized
ducks presented anti-preS antibody titers ranging from 400 to 12,800 (Table 1).
To define whether maternal antibodies from DNA-immunized ducks can
protect offspring against viral infection, progeny ducklings
received a
high-titer (5 × 10
7 vge) challenge with DHBV and the
viremia of individual ducks
was monitored for 17 days. As summarized in
Table
1, all six
progeny ducklings that hatched from control
pCI-immunized ducks,
which had no detectable anti-preS antibodies,
became infected
(Table
1). Out of eight ducklings hatched from
pCI-preS/S-immunized
ducks, two ducklings which had anti-preS antibody
titers of
3,200
at hatch became infected, as evidenced by detection
of DHBV DNA
in their sera. By contrast, six out of six ducklings with
anti-preS
antibody titers of
6,400 at hatch were protected against
DHBV
challenge.
In summary, this study demonstrates for the first time that following
DNA vaccination of laying ducks against large hepadnavirus
envelope,
specific, highly neutralizing, and protective antibodies
are
transferred from the sera of immunized ducks via the egg yolk
to the
progeny of vaccinees. The plasmid used in this study bears
the gene
that expresses the DHBV large envelope protein, which
contains major
virus neutralization epitopes (
17). Interestingly,
as shown
here, DNA immunization of ducks with as little as 100
µg of plasmid
allowed us to generate large amounts (i.e., 60 to
100 mg from each egg)
of highly neutralizing and purified IgY.
In addition, anti-preS egg
yolk antibodies were long-lasting,
with little egg-to-egg titer
variation. Therefore, our study demonstrates
that eggs from genetically
immunized ducks may represent a potent
source of DNA-designed
antibodies specific to viral antigens.
In this regard, DNA immunization
is known to induce a robust immune
response in murine models against
viral antigens (
7). However,
the low antibody yield obtained
in mice hampered the use of this
approach as a source of antiviral
antibodies. In contrast, daily
egg collection from DNA-immunized ducks,
or from chickens, which
are reared more easily, allows the rapid and
noninvasive production
of considerable amounts of virus-specific IgY
antibodies. Such
egg yolk IgYs are known to be valuable
immunodiagnostic reagents,
since they do not react with mammalian
globulins due to structural
differences (
4,
11,
12). Because
mutations can easily be
introduced into plasmid DNA by site-directed
mutagenesis, genetic
vaccination of breeding avians may permit the
rapid generation
of IgY antibodies specific to mutated viral proteins.
Such custom-designed
IgY antibodies may represent interesting tools for
the detection
of the growing number of human HBV variants which escape
actual
immunodiagnostic tests (
9).
Importantly, we show here that hatchlings from DNA-immunized ducks were
protected against DHBV infection. This is of particular
interest,
since, to date, the protective effect of maternal antibodies
transmitted from DNA-vaccinated avians to offspring has not yet
been
investigated. In this regard, conventional protein vaccination
of
breeding avians is known to be a useful approach for conferring
to
their hatchlings passive immunity to viral infection, as was
documented
for avian reovirus (
14). Our results are promising
and
suggest that genetic immunization may have a great potential
for
improving early life protection of domestic avian species
against
different viral
infections.
The protective efficacy of a DNA vaccine against HBV envelope proteins
has been studied with two chimpanzees and showed partial
protection,
since limited virus replication occurred after challenge
(
15). However, the effectiveness of maternal antibodies
induced
by a DNA vaccine for offspring protection against hepadnavirus
infection has not yet been investigated. Our study demonstrates
that
DNA immunization of breeding ducks with the plasmid bearing
the gene
encoding DHBV L envelope protein confers a significant
level of
protection, since 75% of hatchlings resisted DHBV challenge.
In
addition, we have challenged ducklings with a particularly
large amount
of virus, i.e., 5 × 10
7 virus genomes, whereas
neonatal ducklings, known to be highly
sensitive to DHBV, can be
infected with as little as 1 virus genome
(
10). The outcome
of challenge was correlated with anti-preS
antibody titers at hatch.
Thus, only two ducklings with low anti-preS
antibody titers at hatch
(
3,200) became infected whereas all
six ducklings with higher
anti-preS antibody titers were protected
against DHBV challenge. A
strong correlation between anti-HBV
envelope antibody titers and the
efficiency of protection has
also been described for chimpanzees
(
6). These observations
are consistent with the mechanism of
antibody-mediated antiviral
protection, which was analyzed in detail by
Bachmann and coworkers
(
1).
Taken together, our results demonstrate that genetic immunization
against L hepadnavirus envelope protein is capable of inducing
maternal
antibodies which are vertically transmitted and which
confer protection
to progeny against virus infection. Interestingly,
the maternal
transmission of anti-human immunodeficiency virus
antibodies in
response to DNA vaccination has been recently reported
for pregnant
chimpanzees by Bagarazzi et al. (
2), and those
authors have
suggested the usefulness of genetic immunization
for the reduction of
mother-to-fetus virus transmission. Whether
this approach can be
applied for the control of HBV transmission
requires further
study.
| |
ACKNOWLEDGMENTS |
This work was supported by grant 9720 from the ARC. Christine
Rollier is a fellow of the Ministère de l'Education Nationale de
la Recherche et de la Technologie.
We thank Christelle Deleage for excellent technical assistance. We are
grateful to Alan Kay for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Recherche sur les Virus des Hépatites et les Pathologies
Associées, INSERM U271, 151 cours Albert Thomas, F-69424 Lyon
Cedex 03, France. Phone: 33 4 72 68 19 81. Fax: 33 4 72 68 19 71. E-mail: cova{at}lyon151.inserm.fr.
| |
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Journal of Virology, May 2000, p. 4908-4911, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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