Transplacental Genetic Immunization After Intravenous
Delivery of Plasmid DNA to Pregnant Mice1
Kenji Okuda,2* Ke-Qin Xin,* Atsushi Haruki,† Susumu Kawamoto,* Yoshitsugu Kojima,*
Fumiki Hirahara,† Hidechika Okada,‡ Dennis Klinman,§ and Kenji Hamajima*
A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to
the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant micewith liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding
Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the
fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationicliposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring ofvaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, andwere protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong
in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers
confer the Ag-specific immunity to their progeny. The Journal of Immunology, 2001, 167: 5478 –5484.

M

ost vaccines intended for human use are administered
to infants and children. Due to the immaturity of theirimmune system, newborns exposed to foreign Ags are
at risk of developing tolerance rather than immunity (1– 6). For
example, if Ag is administered shortly after birth, forbidden clones
can emerge and induce such tolerance (1– 6). A number of factors
influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as
well as the age of the host (7, 8). Over the past decade, there has
been considerable interest in the use of DNA vaccines to prevent
infection by pathogenic viruses, bacteria, and parasites, with phase
I clinical trials being initiated against malaria, HIV-1, and hepatitis
B virus. These safety studies enrolled normal adults, but if successful, could broaden to include women of child-bearing age andnewborns. Yet there are reports of neonatal tolerance developing
when a DNA vaccine encoding the circumsporozoite Ag of malaria was injected into newborn mice (7). There is also a reportshowing that plasmid DNA can be transmitted to fetuses throughthe placenta, and that the protein encoded by such a plasmid issynthesized by fetal cells (9).
In the present study, we confirmed that plasmid DNA administered to pregnant mice can reach the fetus through the placenta.
This was true both of DNA vaccines encoding the env and gag
genes of HIV-1 as well as those encoding the influenza virus hem-

Departments of *Bacteriology and †Gynecology, Yokohama City University Schoolof Medicine, Yokohama, Japan; ‡Department of Molecular Biology, Nagoya City
University School of Medicine, Nagoya, Japan; and §Center for Biologic Evaluation
and Research/Food and Drug Administration, Bethesda, MD 20892
Received for publication May 21, 2001. Accepted for publication August 31, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by a grant-in-aid from the Ministry of Education, Sciences, Sports, and Culture of Japan and the Japan Health Sciences Foundation(K-1027, SA24713).
2

Address correspondence and reprint requests to Dr. Kenji Okuda, Department of Bacteriology, Yokohama City University School of Medicine, 3-9 Fukuura Kanazawa-ku,
Yokohama 236-0004 Japan. E-mail address: kokuda@med.yokohama-cu.ac.jp

Copyright © 2001 by The American Association of Immunologists

agglutinin (HA),3 matrix (M), and nucleoprotein (NP) genes. Analysis of the immune response of offspring whose mothers were
immunized with the influenza DNA vaccine indicates that theseprogenies had enhanced level of protection against the same virus
infection.

Materials and Methods
Animals
We used 6- to 10-wk-old BALB/c female mice purchased from Japan SLC(Shizuoka, Japan). All mice were allowed free access to sterile food andwater.

Viral protein expression plasmids and Abs
pCMV160IIIB encoding the env gene of HIV-1 strain IIIB has been described in detail in our previous report (10). pCMVgag was constructedfrom the gag gene of HIV-1 strain IIIB using the same plasmid vector ofpCMV160IIIB plasmid. In addition, a plasmid DNA preparation used in
the present study contained liposomes (11).
A pME18S-M expression plasmid was constructed with the pME18S
expression vector into which M region cDNA from influenza virus strain
A/PR/8/34 (H1N1) had been inserted (12). pME18S empty vector was used
as a control plasmid for A/PR/8/34 challenge. The expression of the proteins was confirmed by Western blot analysis (9). DNA vaccines of NP(A/pCMV-V1NP) and HA (V1J-HA(PR8)) (13, 14) genes of the A/PR/
8/34 strain (15–17) were kind gifts from J. J. Donelly and D. Montgomery,Merck Research Laboratory (West Point, PA). Anti-HA (C 179) and antiM1 mAbs (mAb, C111) were purchased from Takara Biomedicals (Kyoto,
Japan). To confirm that plasmid DNA was transferred through the placenta,
a lacZ expression plasmid containing a chicken ␤-actin promoter was
also used.

Virus
Mouse-adapted influenza A/PR/8/34 (H1N1) viruses were used in thisstudy. Viruses were harvested from infected Maudin-Darby canine kidneycells and titrated according to the plaque formation method.

DNA immunization
Mothers were injected i.v. with DNA vaccine before or after coitus. Preparations containing various doses of the DNA vaccine were encapsulated
3
Abbreviations used in this paper: HA, hemagglutinin; DTH, delayed-type hypersensitivity; FISH, fluorescence in situ hybridization; M, matrix; NP, nucleoprotein;p.c., postconception.

0022-1767/01/$02.00

The Journal of Immunology

5479

into liposomes (11). Briefly, a mixture of 3␤-[N⬘-(N⬘-dimethylaminoethane) carbamoyl] cholesterol and dioleoylphosphatidylethanolamine inchloroform was dried, vacuum desicated, resuspended in sterile HEPES
buffer (pH 7.4), and used for the cationic liposomes. Before administration,
an appropriate amount of DNA in 0.15 mol PBS (pH 7.2) was mixed withthe liposome solution at a volume ratio of 17:3. The pregnant mice werei.v. administered the DNA vaccine or empty vector on various days after
postconception (p.c.) to assess immunogenicity. Six weeks after birth, theiroffspring were injected i.m. in gastrocnemius muscles with 20 –50 ␮g ofthe same expression plasmid or the empty vector. For the influenza virus
challenge experiment, 50 ␮g each of plasmids expressing influenza HA and
NP protein was administered into pregnant mice by i.v. route. The sameamount of DNA plasmids were administered to offspring by intranasal
route.

Slot blot analysis
The conventional method for detecting DNA was performed using slot-blot
analysis described elsewhere (9).

Fluorescence in situ hybridization (FISH) analysis
FISH analysis was performed essentially according to the method of ElNaggar et al. (18). A 564-bp HIV env region fragment (nt 1569 –2133)
amplified from pCMV160IIIB plasmid (10) was labeled with digoxigenin11-dUTP by nick translation and used for a hybridization probe. The tissue
samples were taken from mice to which 50 ␮g HIV plasmid pCMV160IIIB(10) or influenza plasmid V1J-HA(PR8) (14, 15) with liposomes was administered and sliced to prepare histological examination. The samples,
which were deparaffinized in xylene and ethanol, were denatured and hybridized with digoxigenin-labeled probes using previously describedmethod (18). After hybridization, the slides were washed and stained withan antidigoxigenin rhodamine (a red fluorochrome; Boehringer Mannheim,
Mannheim, Germany). The slides were counterstained with 4,6-diamino2-phenylindole dihydrochloride. Images were made with a Nikon SA fluorescence microscope (Nikon, Tokyo, Japan) and a charge-coupled device camera interfaced with a Cyto Vision (Applied Imaging, Sunderland, U.K.).

RT-PCR
Total RNA was isolated using TRIzol (Life Technologies, Grand Island,NY) by means of previously described methods (19, 20). Briefly, quick
frozen samples were thawed and extensively treated with DNase. ThenRNA was converted to cDNA using a GeneAmp RNA PCR kit(PerkinElmer, Norwalk, CT). The total RNA was reverse transcribed withrandom hexamers and Moloney murine leukemia virus reverse transcriptase. As a control experiment, we tested RNase treatment before reverse
transcription. For detecting the HIV-1 env gene, cDNA was amplified in a
30-cycle PCR using primers GGAGCAGCAGGAAGCACTAT and TCCCTGCCTAACTCTA. The primers for nested PCR were TGCTGAGGGCTATGA and GCCTCCTACTATCATTATG. For detecting ␤-actin gene, theprimers TGGAATCCTGTGGCATCCATGAAAC and TAAAACGCAGCTCAGTAACAGTCCG were used.

FIGURE 1. Gene expression of lacZ in mouse fetus. A and B, Stained
tissue of a fetus cut in two, whose mother received 30 ␮g of lacZ plasmidwith liposomes at day 9.5 postcoitus (p.c.); C and D, stained tissue of a
fetus whose mother received 30 ␮g of empty plasmid with liposomes atday 9.5 p.c.

ELISA
ELISA was used for titration of serum Ag-specific IgG, IgG1, IgG2a, andIgM responses. Offspring of both immunized and nonimmunized mothers
were bled at 7 days after i.m. immunization with various DNA vaccines, as
described elsewhere (26). A total of 10 ␮g/ml gp160 protein, HGP-30
peptide, rNP protein, M1 synthetic peptides, or heat-killed A/PR/8/34 virus
was coated on 96-well microplates (Nunc, Roskilde, Denmark); after
blocking with 3% BSA in PBS, serially diluted antisera were added andincubated at 37°C for 2 h. Peroxidase-conjugated goat anti-mouse IgG(Organon Teknika, West Chester, PA) was used as the secondary Ab; thenRecombinant proteins and peptides
The DNA fragments encoding almost the full length of the influenza M andNP genes were amplified from pME18S-M and A/pCMV-V1NP by PCR.
The PCR products were inserted into pGEX-3X (Pharmacia Biotech, Uppsala, Sweden) by directional cloning. pGEX-M and pGEX-NP were transformed into Escherichia coli JM 101. Purified influenza M gene productand NP protein were obtained using a bacterial expression system. Recombinant gp160 from HIV-1IIIB was obtained from MicroGene System(Meriden, CT). HGP30 peptide (YCVHQRIEIKDTKEALDKIEEEQNKSKKKA) was used for detection of the HIV-1 gag immune response (21,22); and influenza M1 peptide (KAVKLYRKLKRE) was used as an Ag for
ELISA. We also included mice that were i.m. injected inactivated influenza
A/PR/8/34 as a control Ag. For the CTL assay, we used HIV-1 V3 peptide(RGPGRAFVTI) (23) to synthesize the Ag as well as the peptide
ALVEADVA of sperm whale myoglobin (24) as a control peptide.

Delayed-type hypersensitivity (DTH) response
Seven days after i.m. immunization with 20 ␮g of the same expression
plasmids as received by their mothers, a total of 25 ␮l of PBS containing
5 ␮g of gp160, HGP-30 peptide, influenza M gene product, or heat-killed
A/PR/8/34 virus was injected into the right footpad of immunized offspring. After 24 h, the extent of footpad swelling was measured by previously described method (25).

FIGURE 2. FISH analysis. Vertebra of a fetus whose mother received
HIV plasmid pCMV160IIIB (A) or influenza plasmid V1J-HA(PR8) (B)with liposomes at day 9.5 p.c. These samples were reacted with HIV env
region fragment labeled with digoxigenen-11-dVTP, followed by staining
with an antidigoxigenin rhodamine. Red fluorochrome indicates the presence of HIV-IIIB DNA.

5480

TRANSPLACENTAL IMMUNIZATION
Table I. Ab response of the progeny after i.v. injection of pregnant mice with expression plasmidsa
Reciprocal Log2 Titer
Immunogens

gp160

HGP-30 peptide

A/PR/8/34

NP protein

M1 peptide

pCMV160IIIB (50 ␮g)
pCMV160IIIB (10 ␮g)
pCMV160IIIB (1 ␮g)
Empty vector (50 ␮g)
pCMV160IIIB (50 ␮g)c
(without liposome)pCMV gag (50 ␮g)
Empty vector (50 ␮g)
pCMV gag (50 ␮g)c
(without liposome)pCMV gag pol (50 ␮g)
Empty vector (50 ␮g)
V1J-HA(PR8) (50 ␮g)
Empty vector (50 ␮g)
A/pCMV-V1NP (50 ␮g)
Empty vector (50 ␮g)
pME18S-M (50 ␮g)
Empty vector (50 ␮g)

9.6 ⫾ 1.0
6.5 ⫾ 1.1b
5.7 ⫾ 0.4
4.3 ⫾ 0.7

⬍2
⬍2
⬍2
NT

⬍2
⬍2
NT
NT

NT
NT
NT
NT

NT
NT
NT
⬍2

7.2 ⫾ 1.3b
⬍2
⬍2

⬍2
10.8 ⫾ 2.0b
4.0 ⫾ 0.4

⬍2
NT
NT

NT
NT
NT

⬍2
NT
NT

⬍2
⬍2
⬍2
NT
NT
NT
NT
NT
NT

7.4 ⫾ 1.8b
9.0 ⫾ 1.4b
4.8 ⫾ 0.3
⬍2
⬍2
⬍2
⬍2
NT
NT

NT
NT
NT
10.7 ⫾ 1.7b
5.3 ⫾ 1.0
NT
NT
NT
NT

NT
⬍2
⬍2
NT
NT
6.7 ⫾ 0.9b
4.2 ⫾ 0.6
NT
NT

NT
NT
NT
2.2 ⫾ 0.3
1.9 ⫾ 0.4⬍2NT
8.7 ⫾ 1.4b
4.2 ⫾ 0.3ba

At day 9.5 p.c., pregnant BALB/c mice were i.v. injected with each expression plasmid, or empty vector, and liposomes.
Significant difference ( p ⬍ 0.05) from the empty vector control group. NT, not tested. Two other separate experiments showed similar results.
Fifty milligrams of pCMV160IIIB was i.v. injected without liposomes. Six weeks after birth, their progeny were immunized i.m. with 50 ␮g of thesame expression plasmids without liposomes as received by mothers. The offspring whose mothers received empty vector were also immunized with theexpression plasmids of the same group. After 7 days, blood was collected and the Ab titers were detected by ELISA using each gp160, HGP-30 peptide,
heat-killed A/PR/8/34 virus, NP, or M1 peptide. Data represent means ⫾ SE of five to eight mice.
bcthe plates were stained with 3,3⬘,5,5⬘-tetramethylbenzine (DAKO, Carpenteria, CA). For Ag-specific IgG1, IgG2a, and IgM responses, HRP-coupled
anti-mouse IgG1, IgG2a, or IgM (Organon Teknika) was used as the secondary Ab. Specific Ab titers were expressed as the reciprocal value of the
final dilution, which gave an OD (A415) of ⭌ 0.2 OD units compared witheach preimmunized sample.

Cytokine ELISPOT assay
The cytokine ELISPOT assay was performed with minor modifications, as
previously described (26, 27). Briefly, 96-well microplates (MAIPS4510;
Millipore, Bedford, MA) were coated with anti-mouse IFN-␥ rat mAb (BDPharMingen, San Diego, CA), and after adding cells isolated from thespleen 7 days after immunization, plates were incubated in a 5% CO2
atmosphere at 37°C with or without 10 ␮g/ml V3 peptide. After a 48-h
culture, plates were washed and incubated again for 1 h with biotinylated
anti-mouse IFN-␥ mAb (BD PharMingen). Then, after peroxidase staining,
the spots in each well were counted, using the dilution to calculate the total
number of cytokine-secreting cells utilizing a computer-assisted video image system (Zeiss, Oberkochen, Germany).

HIV-1-specific CTL response
Mother mice that received pCMV160IIIB were immunized with 50 ␮g ofthe same plasmid with liposomes, and spleen cells were collected. The

details of the CTL assay have been described previously (28). We also
obtained spleen cells from offspring whose mothers were injected with theempty vector. Then 1 ⫻ 106 lymphoid cells were restimulated in vitro in
the presence of the same amount of irradiated (30 Gy) syngenic spleen cellswith 5 ␮g/ml HIV-1 V3 peptide RGPGRAFVTI (29, 30), a known CTL
epitope of HIV-1IIIB. After a 5-day culture, the cytotoxic activity of thesespleen cells was measured by a 6-h 51Cr release assay using V3 or myoglobin peptide-pulsed target P815 (H-2d) cells.

Virus challenge
Under light diethylether anesthesia, the offspring were simultaneously
infected with virus at day 10 after immunization with the same plasmid
DNA as that administered to their mothers. Five lethal doses (LD50) of
influenza A/PR/8/34 (H1N1) in 30 ␮l of PBS were administered by the
intratracheal route using a 24-gauge stainless steel animal feeding tube
(Popper & Sons, New York, NY). The mortality rate was determined
after 20 days.

Statistical analysis
Statistical analysis for comparison of two groups was conducted using an
unpaired t test or one-way factorial ANOVA for distribution parameters.
Significance was defined as p ⬍ 0.05 in both analyses.

Table II. Effect of timing of DNA vaccination of mothers on Ab responses of their progenya
Timing of DNA Vaccination into
Group

1
2
3
4
5
6
7

Mothers

2 wk before coitus (pCMV160IIIB)
0–3 wk p.c. (pCMV160IIIB)
5–8 wk p.c. (pCMV160IIIB)
9–11 wk p.c. (pCMV160IIIB)
12–18 wk p.c. (pCMV160IIIB)
10 wk p.c. (empty vector)
10 wk p.c. (empty vector)

Reciprocal Log2 Titer
Progeny (6 wk)

gp160 protein

pCMV160IIIB
pCMV160IIIB
pCMV160IIIB
pCMV160IIIB
pCMV160IIIB
pCMV160IIIB
Empty vector

4.1 ⫾ 1.2
6.0 ⫾ 0.7
8.2 ⫾ 0.8*
10.9 ⫾ 1.6*
10.5 ⫾ 1.2*
4.2 ⫾ 1.5
2.4 ⫾ 0.3aMice at various stages of pregnancy were i.v. injected with 50 ␮g of pCMV160IIIB, or empty vector, with liposomes. In
one group, the mothers were i.v. administered pCMV160IIIB at 2 wk before coitus. Six weeks after birth, all offspring except
those of group 7 were immunized i.m. with 50 ␮g of the same expression plasmids as received by their mothers. In a control
group, mothers and offspring were injected i.m. with empty vector only. After 7 days, blood was collected and the Ab titers weredetected by ELISA using gp160.
b
Significant difference compared with group 6 ( p ⬍ 0.05). Data represent means ⫾ SE of four to eight mice.

The Journal of Immunology

5481

Table III. Types of Ab response of offspring after i.v. injection of pregnant mothers with pCMV160IIIB or pCMV-V1NPa

Reciprocal Log2 Titer
Immunization of

IgG1

IgG2a

IgM

Group

Pregnant
mothers

Progeny

Beforeimmunization

After
immunization

Beforeimmunization

After
immunization

Beforeimmunization

After
immunization

1
2
3
4
5

pCMV160IIIB
Empty vectorpCMV-V1NP
Empty vector
Empty vector

pCMV160IIIB
pCMV160IIIB
pCMV-V1NP
pCMV-V1NP
Empty vector

4.1 ⫾ 0.6
⬍2
4.5 ⫾ 0.8
⬍2
⬍2

10.3 ⫾ 1.3b
5.4 ⫾ 0.7
8.9 ⫾ 2.4b
5.9 ⫾ 0.9
⬍2

3.0
⬍2
2.7
⬍2
⬍2

6.0 ⫾ 0.9
5.3 ⫾ 1.2
5.5 ⫾ 0.7
5.0 ⫾ 0.7
⬍2

4.3 ⫾ 1.1
⬍2
4.6 ⫾ 0.4
⬍2⬍2

6.7 ⫾ 1.8
5.2 ⫾ 0.4
5.0 ⫾ 0.4
5.3 ⫾ 0.2⬍2aAt day 9.5 p.c., pregnant BALB/c mice were i.v. injected with 50 ␮g of pCMV160IIIB, pCMV-V1NP, or empty vector, with liposomes. Six weeks after birth, progeny ofgroups 1 and 2 were immunized i.m. with 50 ␮g of pCMV160IIIB. In groups 3 and 4, offspring were injected i.m. with pCMV-V1NP with liposomes. Two days before
immunization, the blood samples were collected (before immunization). After 7 days, blood samples were collected (after immunization) and Ab titers were detected by ELISA
using gp160 or NP, respectively. Data represent means ⫾ SE of six to eight mice.
b
Significant difference ( p ⬍ 0.05) from the empty vector control.

ted to the fetuses (Fig. 2). We found abundant plasmid DNA in
spleen, liver, lung, and other tissues (data not shown). Further
evidence of extensive plasmid transfer was provided by RT-PCR
analysis of tissues from newborn mice. mRNA of the HIV-1 env
gene could be detected in mice whose mothers had received the
DNA vaccine from 5.5 to 17.5 days p.c. (data not shown).

Results
Gene transfer into fetuses
Initial studies examined whether DNA plasmids could be transmitted through the placenta of pregnant mice. To evaluate plasmid
uptake and expression in fetal tissue, a plasmid expressing the lacZ
gene was used. To increase the uptake of this plasmid, it was
liposome encapsulated before i.v. delivery (7). Tissues from newborn mice from mothers injected with the lacZ plasmid and liposomes 9.5 days p.c. showed strong expression of that gene (Fig. 1).
Of particular interest was the intense staining in the umbilical region of the fetuses.
Slot-blot analysis of offspring whose mothers had received liposome-encapsulated plasmid DNA-encoding genes from HIV
was performed as previously described (9). There was extensive
evidence of plasmid DNA reaching these fetuses of mothers injected at 9.5 and 14.5 days of gestation (data not shown). In a
separate experiment using densitometric analysis, when mice immunized with pCMV160IIIB and liposomes 9.5 days p.c. werecompared with those immunized with pCMV160IIIB only, it was
found that the uptake in those treated with liposomes was 3.2 times
that in those that were not so treated. Examination by the FISH
method confirmed that abundant plasmid DNA had been transmit-

Immune responses of mice born from plasmid DNA-vaccinated dams
The offspring of mice vaccinated while pregnant with one of several different DNA plasmids were immunized at 6 wk of age withthe same plasmid. Ab titers among offspring of vaccinated mothers
were significantly higher than those of mothers injected with vector alone (Table I). Improved Ab responses were seen for a variety
of epitopes encoded by HIV-1 and influenza genes. The Ab titersof mice at 6 wk of age whose mothers received plasmid DNA were
below 2 when we tested ELISA using each corresponding Ag (datanot shown). The timing of maternal DNA vaccination on the immune response of offspring was then examined. Mothers were immunized either 2 wk before being mated, or at various periods p.c.
At 6 wk postbirth, the offspring were vaccinated and their Ab
response was tested after 7 days. Vaccination before mating had no

Table IV. Ag-specific DTH responses of progeny after i.v. injection of pregnant mice with various expression plasmidsa
Footpad Swelling Response (⫻10⫺3 cm)
gp160

HGP-30

A/PR/8/34

M1 peptide

Immunogens

Beforeimmunization

After
immunization

Beforeimmunization

After
immunization

Beforeimmunization

After
immunization

Beforeimmunization

After
immunization

pCMV160IIIBb (50 ␮g)
pCMV160IIIB (50 ␮g)
pCMV160IIIB (10 ␮g)
pCMV160IIIB (1 ␮g)
Empty vector (50 ␮g)
pCMV gag (50 ␮g)
Empty vector (50 ␮g)
V1J-HA(PR8) (50 ␮g)
Empty vector (50 ␮g)
pME18S-M (50 ␮g)
Empty vector (50 ␮g)

3.2 ⫾ 1.0
4.7 ⫾ 1.2
4.6 ⫾ 0.2
4.9 ⫾ 0.3
2.5 ⫾ 0.4
NT
NT
NT
NT
NT
NT

7.6 ⫾ 1.1
15.3 ⫾ 3.1c
9.8 ⫾ 0.4c
10.7 ⫾ 0.6c
10.4 ⫾ 1.9
2.4 ⫾ 0.3NT
2.2 ⫾ 0.2NTNT
0.9 ⫾ 0.1

NT
NT
NT
NT
NT
3.5 ⫾ 0.5
2.0 ⫾ 0.4
NT
NT
NT
NT

NT
NT
NT
NT
NT
14.6 ⫾ 3.2c
9.2 ⫾ 1.3
2.5 ⫾ 0.6
1.8 ⫾ 0.3NT
2.2 ⫾ 0.6

NT
NT
NT
NT
NT
NT
NT
4.1 ⫾ 0.6
2.3 ⫾ 0.5NTNT

2.0 ⫾ 0.2
1.6 ⫾ 0.7
2.2 ⫾ 0.4
1.4 ⫾ 0.3
1.7 ⫾ 0.5
2.3 ⫾ 0.6
5.2 ⫾ 0.6
14.2 ⫾ 1.7c
6.5 ⫾ 1.0
1.9 ⫾ 0.3
2.1 ⫾ 0.3NT
2.6 ⫾ 0.6
NT
NT
NT
NT
NT
NT
NT
4.2 ⫾ 0.8
1.4 ⫾ 0.4NT
1.8 ⫾ 0.4
NT
NT
NT
NT
NT
1.7 ⫾ 0.5
1.3 ⫾ 0.2
14.8 ⫾ 2.7c
6.0 ⫾ 0.4a
At day 9.5 p.c., pregnant BALB/c mice were i.v. injected with each expression plasmid, or empty vector, and liposomes. Six weeks after birth, their progeny were immunizedi.m. with 20 ␮g of the same expression plasmids with liposomes as their mothers received (after immunization).
b
About 2 wk before coitus, the mothers were administered 50 ␮g of pCMV160IIIB and liposomes, and their offspring were immunized i.m. with 20 ␮g of pCMV160IIIB.
After 7 days, these mice were challenged with 5 ␮g of gp160, HGP-30 peptide, heat-killed A/PR/8/34 virus, and M1 peptide, respectively. Data represent means ⫾ SE of fiveto nine mice.
c
Significant difference ( p ⬍ 0.05) from the empty vector control group. Two other separate experiments showed similar results.

5482

TRANSPLACENTAL IMMUNIZATION
Table V. ELISPOT analysis of IFN-␥-producing spleen cells from DNA-vaccinated mice whose mothers
had been injected with the same vaccine during pregnancya
Immunogen Administered toPregnant mother

Progenies

IFN-␥-Producing Cells(spot/106 spleen cells)Group 1: pCMV160IIIB
Group 2: pCMV160IIIB
Group 3: Empty vector
Group 4: Empty vector
Group 5: Nonimmune

pCMV160IIIB
Nonimmune
pCMV160IIIB
Empty vector
Nonimmune

42.6 ⫾ 9.6b,c
20.6 ⫾ 6.7c
28.2 ⫾ 3.5*
16.7 ⫾ 6.3
13.9 ⫾ 5.9a
At day 9.5 p.c., pregnant BALB/c mice were i.v. injected with 50 ␮g of pCMV160IIIB, or empty vector, with liposomes.
Six weeks after birth, the offspring were immunized i.m. with 50 ␮g of the same plasmid or empty vector as received by theirmothers. After 7 days, spleen cells were collected and cocultured with V3 peptide for 2 days. Data represent means ⫾ SE of six
to eight mice.
b
Significant difference ( p ⬍ 0.05) from the empty vector control.
cSignificant difference between these two. The data of two other experiments showed similar results.

effect on the immune response of the litter (Table II). By comparison, significantly Ag-specific responses were observed in the offspring of mice vaccinated from 5 to 18 days p.c. In general, thelater vaccination was delayed p.c., the stronger the immune response mounted by the immunized offspring.
The isotype of Ab produced by vaccinated offspring of immunized mothers was examined. There was a significant increase in
Ag-specific IgG1, but not IgG2a or IgM titers in these mice (Table
III). To provide a measure of Ag-specific T cell immunity, offspring of DNA-vaccinated mothers were immunized at 6 wk of age
with the same vaccine, and then challenged in the hind footpad 1
wk later with 5 ␮g vaccine-encoded Ag. Significant enhancementof Ag-specific footpad-swelling response was observed in the offspring of dams vaccinated with 50 ␮g plasmid DNA (Table IV).
Lower doses of maternal vaccination were less effective. Of note,vaccinating either the father or mother before conception did notalter the immune response of the offspring (data not shown). These
results suggest that the offspring of vaccinated dams develop improved DTH responses as well as Ab responses by this technique.
Confirming the antigenic specificity of this effect, mice challenged
with the wrong Ag manifest normal rather than heightened footpad-swelling response (Table IV).
ELISPOT analysis using spleen cells from immunized offspringof vaccinated mothers was performed (Table V). When stimulated
in vitro with vaccine-encoded Ag, a significant increase in thenumber of spleen cells secreting IFN-␥ was observed.
The CTL response was then investigated and found to be enhanced in offspring whose mothers had received DNA vaccine. In
mice of mothers treated with DNA vaccine, DNA plasmids weretransmitted into the fetuses through the placenta, resulting in the
induction of a stronger CTL response (Fig. 3).

their offspring to develop protective immunity against viral challenge postvaccination.

Discussion
Three independent techniques were used to establish that plasmid
DNA administered to pregnant mice could reach the fetus. First,
administration of a ␤-galactosidase-encoding plasmid allowed forthe direct identification of protein expression in neonates. Although protein levels were low (they were undetectable by Western blot analysis), clear detection of multiple organs and intense
staining of the placenta were consistent with transplacental migration of the plasmid. Second, RT-PCR allowed identification ofmRNA encoded by the DNA vaccine in fetal tissues, especiallythose from mothers injected after day 9.5 p.c. DNA vaccination in
the precoital period or before day 3 of pregnancy did not result in
detectable fetal transmission of the plasmid, presumably due toChallenge test with influenza virus
To examine the immunoprotective effect of maternal vaccination
with a DNA vaccine against influenza virus, offspring were immunized with 50 ␮g of the same vaccine at 6 wk of age. Sevendays later, they were challenged with influenza virus A/PR/8/34.
Whereas few normal mice survived challenge, ⬎70% of the offspring of vaccinated mothers survived (Fig. 4).
The timing of maternal DNA vaccination on the capacity ofoffspring to develop protective immunity was then examined.
Whereas ⬍20% of normal vaccinated mice (and offspring of
mother vaccinated 20 days before mating) survived challenge,⬎50% of the offspring of immunized mothers survived (Table VI).
These findings indicate that immunization of mothers with a
DNA vaccine against the influenza virus improves the ability of

FIGURE 3. CTL response of immune BALB/c mice, whose mothers
had been administered with the same DNA vaccine. Day 9.5 p.c. pregnant
BALB/c mice were i.v. injected with 50 ␮g of pCMV160IIIB with (䡺) orwithout (Œ) liposomes. Six weeks after birth, some offspring were immunized i.m. with 50 ␮g of pCMV160IIIB. Progeny of mothers that received
only empty vector were immunized i.m. with pCMV160IIIB (F). Normal
nonimmune mice were used as a control group (f). After 7 days, spleencells were collected and cocultured for another 5 days with V3 peptidepulsed P815 cells. As a control, P815 cells pulsed with the myoglobin
peptide ALVEADVA were also used (E). Data represent means ⫾ SE offour to six mice. Spleen cells from mice immunized twice with 20 ␮g ofmyoglobin with CFA lysed 42.3% of the myoglobin-pulsed target cells at
an E:T ratio of 80:1.

The Journal of Immunology

5483

FIGURE 4. Protection of offspring against alethal A/PR/8/34 influenza virus challenge. Day
9.5 p.c. pregnant BALB/c mice were i.v. injectedwith 25 ␮g each of pME18S-M and pCMVV1NP with (Œ) or without (E) liposomes. Six
weeks after birth, their offspring were immunized i.m. with a total of 50 ␮g of the same plasmid DNA with liposomes. In one group, offspring of mothers that had received DNA
vaccine with liposomes were not administeredvaccine (〫). In another group, the mother andprogenies received only empty vector (‚). Nonimmunized normal mice were used as the othercontrol (f). After 7 days, all mice were challenged with 5 LD50 A/PR/8/34 virus. The percentage of survival of these mice was studied foranother 15 days. n, Number of mice.

immaturity of the placenta at this early stage (Table II). Third, the
FISH method was used to directly detect plasmid DNA in fetal
tissue (Fig. 2). Our results confirm and extend the previous finding(9) that a ␤-galactosidase plasmid can be transmitted through theplacenta to the fetus in mice. Of particular importance, we established that such transplacental transfer influences the recipients’
subsequent capacity to mount an immune response against theplasmid-encoded Ag. This was manifested both by improved humoral and cellular immunity (Tables I-V) and higher levels of
pathogen-specific protection (Fig. 4 and Table VI).
These studies were also performed to test the hypothesis that theadministration of a DNA vaccine during pregnancy may induce
Ag-specific tolerance in the offspring, as suggested by the clonal
selection theory of Burnet (2, 3, 31). Although pregnant mice were
immunized with various doses of several different DNA vaccines,
we found that immunity, but not tolerance, was elicited in the fetus(Tables I-IV). Using this technique, we did not observe Ag-specific immune tolerance in progeny, as reported by Mor et al. (32).
This could reflect our use of a different plasmid (Mor detected
tolerance following neonatal immunization with a plasmid encoding the circumsporozoite protein of malaria), or the very limited
amount of plasmid actually transferred transplacentally. Indeed,
Ichino et al. (7) demonstrated that neonatal tolerance was dose
dependent, and could be reliably induced only when ⬎10 ␮g plas-

mid was injected into newborn mice. This is consistent with otherreports showing that low dose Ag can induce immune responsiveness, while high dose immunization can induce tolerance in young
recipients (8, 33, 34), which might be supporting Burnet’s theory
that high levels of neonatal Ag can trigger clonal deletion (3, 7).
When offspring of vaccinated mothers were immunized at 6 wk
of age with the same DNA vaccine, they manifested significant
anamnestic responses (Table III). Reimmunization was required,however, since transplacental transport of plasmid alone did not
trigger strong immune responses in the newborn, nor provide adequate protection from infection (Fig. 4 and Table VI). Yet reexposure of these mice to vaccine at 6 wk of age elicited a strong,
protective immune response, characterized by Ag-specific Ab, CTL,
and cytokine responses. The administration of DNA vaccine into amniotic fluid induced a high level of protective immunity (35).
We used liposomes throughout our studies, because we could
observe high levels of immune responses (Table I and Fig. 4) when
we added liposomes as an adjuvant. The same observation wasreported previously (9).
Results indicate that the transmission of serum IgG from immunized mothers to offspring seems not to have a major influenceon the production of specific Ab at 6 wk of age. A lack of maternal
contribution to the DTH response of offspring is particularly evident (Table IV). Thus, while we cannot rule out the possibility that

Table VI. The importance of timing of DNA immunization of pregnant mothers against A/PR/8/34 virus
challengea
DNA Vaccination ofPregnant mothers

Group 1
DNA vaccination 20
days before coitus
Day 5.5 p.c.
Day 9.5 p.c.
Day 14.5 p.c.
Day 9.5 p.c.
Group 2
Empty vector day 9.5 p.c.
Group 3Nonimmune control

Survival After Immunization
Progeny

Survivors/total (%)

DNA vaccination

3/19 (15.8)

DNA vaccination
DNA vaccination
DNA vaccination
No vaccination

5/20 (25.0)
11/21 (52.3)b
12/19 (63.2)b
3/18 (16.7)b

DNA vaccination

2/22 (9.1)

Nonimmune

1/19 (5.3)

Pregnant BALB/c mice were i.v. injected with 25 ␮g each of A/pCMV-V1NP plus pME18S-M and liposomes on day 5.5,
9.5, or 14.5 p.c. Six weeks after birth, all offspring were immunized i.m. with 50 ␮g of the same plasmid DNA as received bytheir mothers. After 7 days, these mice were challenged with 5 LD50 of A/PR/8/34 and the percent survival was determined after
another 20 days. Data represent means ⫾ SE of 19 –22 mice.
b
Statistically significant difference ( p ⬍ 0.05) among nonimmune control group. The data of another separate experimentshowed similar results.
a5484
some maternal contribution persisted, only following direct immunization of offspring at 6 wk of age was clear evidence of improved
protective immunity observed (Table VI). However, in the present
study, when DNA vaccine was given to mothers, Ag-specific acquired immunity was induced in their offspring. Therefore, thismethod may be effective in the prevention of pertussis, hepatitis,
and various other infections occurring in infants as well as animals.

Acknowledgments
We thank Akiko Takada for her technical assistance, and Tomoko Takeishi
and Atsuko de la Fuente for their secretarial assistance.

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