This information is current asof March 10, 2010

Intranasal immunization of a DNA
vaccine with IL-12- and granulocytemacrophage colony-stimulating factor(GM-CSF)-expressing plasmids inliposomes induces strong mucosal andcell-mediated immune responses
against HIV-1 antigens

J. Immunol. 1997;159;3638-3647
References
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc., 9650
Rockville Pike, Bethesda, MD 20814-3994.
Copyright ©1997 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from www.jimmunol.org on March 10, 2010

E Okada, S Sasaki, N Ishii, I Aoki, T Yasuda, K Nishioka,
J Fukushima, J Miyazaki, B Wahren and K Okuda

Intranasal lmmunization of a DNA Vaccine with 11-12- andGranulocyte-Macrophage Colony-Stimulating Factor(CM-CSF)-Expressing Plasmids in Liposomes Induces Strong
Mucosal and Cell-Mediated Immune Responses Against HIV-1
Antigens"aEiichi Okada,* Shin Sasaki,*+ Norihisa lshii,* lchiro Aoki,§ Tatsuji Yasuda? Kusuya Nishioka,ll
Jun Fukushima,* Jun-ichi Miyazaki,' Britta Wahren,** and Kenji Okuda2*T

he induction of strong mucosal and cell-mediated immuin connity (CMI)' are important elements to consider
structing an effective HIV- 1 vaccine. We have designed aplasmid DNA vaccine ( I ) to achieve these goals. Our first objective was to stimulate HIV-specific mucosal immunity. Immunologically competent lymphocytes and Abs in mucosal sites form a
critical first line of host defense against local HIV-1 infection. Our
previousresultssuggestedthat
mucosal IgAAbinhibitsHIV-1
replication (2). Although a variety of immunization protocols using HIV-I vaccines have been shown to induce the production ofserum neutralizing Abs after systemic vaccination. immunization
via a systemic route rather poorly induces a secretory IgA immune
response (3-6). Since circulating IgG and IgM only poorly penetrate mucosal surfaces, there is usually little relationship between*Department of Bacteriology. '1 st Department o f Internal Medicine, *Departrnent o f Dermatdogy. and 'Department of Pathology, Yokohama Citv University
School o f Medicine. Yokohama; "Institute of Cellular and Molecular Biology,
Okayarna UniversLty Medical School, Okayarna; "Viral Hepatitis Foundation ofJapan, Tokyo; and 'Department o i Nutrition and PhyslologicalChemistry, Osaka
University Medical School, Osaka, Japan; and **Swedish Institute for Infectious
Disease Control. Karolinska Instlhrtr, Stockholm, Sweden

Received for publlcatlon January9. 1 997. Accepted for publication July 9, 1997.
The costs of publication ot this article were defrayed in part hy the payment ofpage charges. This article must therefore be hereby marked ddwrtkemmt ina t c o r d a x e w < t h18 CJ.S.C. Sectmn 1734 solely to indicate this f x t .

'This work was partially supported by a Grant-in-Ald for Scientiiic Research
fromtheMinistryof
Educatton. Science, Sports. andCulture of Japan (No.
0867011 1 ).

' Address correspondence and reprint requests to Dr. Kenji Okuda, Departmento f Bacteriology, Yokohama City University School
Kanazawa-ku, Yokohama 23h, lapan.

of Medicine, 3-9 Fukuura,'

Abbreviations used in this p q e r : CMI, cell-mediated Immunity; CM-CSF, granulocytdmacrophagecolony-stimulating factor; CT, cholera toxin; DTH, deIayed-type hypersensltivltv; i.n.. intranasal ur intranasally: VJ region, third hvpervariable region.
Copyright 0 1997 by The Arnertcan Association of Immunologists

resistance to mucosally associated infections and levels of circulating IgG or IgM Ab. Tissues of the female reproductive tract,urethra, and several mucosa have cells characteristicof mucosal
effector tissues, including cells that have been shown to produce
mucosal IgA Abs (7). There are some reports that intranasal (in.)
administration of protein or peptide Ags induces both1gA and IgE
Abs ( 5 , 8, Y), which correlates with the activation of the Th2-type
immune response ( 5 ) . However, there have beenonly a few studies
concerning i.n. administration of DNA plasmids (10). The main
purpose of the present study was to determine whether i.n. DNA
vaccination i s capable of inducing a high level of HIV-1-specificmucosal immunities.
Work leading up to the present study involved the constructionof a DNA vaccine from HIV-I em' and rev genes. lmmunization
withthis vaccine resulted in a strong CMI response t o HIV-1
gp160Ag (1). ThisDNAvaccineinduces
Env and atninuteamount of Rev proteins and facilitates CTL production ( I , 1 1 , 12).
Use of cationicliposomes was thought to be advantageousfor
vaccine delivery as they have been reported to be effective adjuvants in immunization (14-16). Therefore, we tested whether i.n.
administration of DNA vaccine with cationic liposomes could activate both mucosal immunity and a strong CMI.
To achieveprotectiveimmunityagainstHIV-1infection,theinduction of high CTL levelshas been showntobeimportant( I 7- 19). TL- I2 enhances various types of CMI by Th I cell activation (20-27). In addition, GM-CSF has beenreported to enhance Ab responses (28). In this regard, we studied whether i.n.
DNA vaccination with 1L-12 andlor GM-CSF expression plasmids
inliposomescouldactivateHIV-specificimmuneresponses.
Coadministration of IL-12 and HIV-1 expression plasmids
byi.n. vaccination significantly enhanced HIV-I-specific CTL production. Coadministration of GM-CSF and HIV- I expression
plasmids enhanced both Thl- and Th2-type responses. Additional(1022.1 767/'37/$02.00

Downloaded from www.jimmunol.org on March 10, 2010

A DNA vaccine constructed with the CMV promoter conjugated to env gp160 and rev geneshas been shown to induce aneffective Thl-type immune response when inoculated via an intramuscular route. In the present study, we obtained high levelsof both humoral andcell-mediatedimmune activity by intranasaladministration ofthis DNA vaccine.The production of
mucosal IgA Ab in feces and vaginal fluid was stimulated significantlyby intranasal DNA administration. This route of administration resulted in a significant level of HIV-1-neutralizing Abs in feces and serum. Cytokine assays revealed that intranasal
administration of this DNA vaccine induces a Th2-type immune response. Interestingly, cationic liposomes greatly enhanced
these activities. Abs against HIV-1 were present for at least 10 mo. Coadministration of the DNA vaccine with IL-12- andgranulocyte/macrophage-CSF-expressing plasmids induced high levels of HIV-specific CTLs and an increase in delayed typehypersensitivity when administered by the intranasal route. These results clearly demonstrate that intranasal administrationof
this DNA vaccine with liposomes,together with IL-12- and/or granulocyte/macrophage-CSF-expressingplasmids,induces astrong level of anti-HIV-I immune response.The
journal of Immunology, 1997, 159: 3638-3647.

3639

The Journal of Immunology

activation o f HIV-specific Th 1 and Th2 cells was observedby mixing these two cytokines. We conclude that i.n. vaccination using
plasmid DNA vaccines encoding HIV Ags plus appropriate cytokine plasmids may be a useful approach for inducing protective
immunity against HIV.

Ai"""
,BamHI

Materials and Methods

12 p40HIV viruses
HIV-Istrains of 111, were kindlydonated by the National Institute\ ofHealth AIDS Research and Reference Reagent Program.

\

Eco F4

AnimalsU

All BALB/c (H-2") mice. 6 to 10 wk old, were purchased from Japan SLC,
Inc.. Hamamatsu, Japan.

CAG prornot er
BamH'

DNA vaccine

EcoR

B
Pvul

CAG promoterpCAGGSIL-4
5.25 Kb

I

Eco A

BamHl

FIGURE 1.

Genetic maps of pCAGGSIL-12 ( A ) and pCAGGSIL-4 ( B ) .

Preparation of liposomes

EL ISAThe detailed method for cationic liposome preparation has
been reported
previously (34). Amixtureof 3p(N(N'.N'-dimethylaminoethane)carbamoyl)cholesterol(DC-Chol)anddioleoylphosphatidylethanolamine(DOPE) in chloroform was dried, vacuum desiccated, and resuspended insterile HEPES buffer (pH 7.8). After hydration, the dispersion was sonicated to form liposomes with an average diameter of I50 to 200 nm. Before
injection, an appropriate amount of DNA plasmids in 0.15 mol PBS. pH
7.2, was mixed with the above liposome solution at a volume ratio of 17:3.
Thehe mixtures were vortexed and injected.

ELISA was performed as described previously (37. 38). First, 96-well microtiterplateswerecoatedwith OI pg/ml of IlIBV3(TRKSIRIQRG
PCRAFVTIGKICN) long peptide (2) or 1 pg/ml of gp160 protein. whichwere donated by the National Institutes of Health AIDS Research andReference Reagent Program. The wells were rinsed with 0.15 M PBS andthen blocked with 3% BSA in PBS for I h. Mouse sera were added andincubated for 1 h at 37°C. Following washing, the bound Ig was characterized using affinity-purified horseradish peroxidase-coupledgoatantimouse IgC + A + M (H + L) + E(PierceChemicals),orhorseradish
peroxidase-coupled goat anti-mouse isotype-specific Abs for IgC1, IgG2a.
and IgM (Southern Biotechnology As5ociates). For the estimation of secretory IgA (s-IgA) Ab against the V3peptide. rabbit anti-rat secretorycomponent (SC) Ab (kindly provided
by Dr. B. Underdown,McMaster
University Medical Center, Ontario. Canada) was alsoused. The titer$ werereported a s the reciprocal number of the final detectable dilution. whichgave anOD,,,, of 20.2 OD units.

Immunizafion and DNA inoculation
Direct inoculation of the DNA vaccine into the gastrocnemius or quadriceps muscles of mice was performed as previously described( I . 35). Mostof the data described in this paper were obtained by immunizing mice inthe gastrocnemius muscles. I n some cases, however, we inoculated into thequadriceps or biceps muscles to compare the immunogenicityof this DNA
vaccine at dilferent sites. No diserences in immune activities have beenreported between these muscles (34). Mice were immunized with a total of
I to IO pg of DNA vaccine in PBS mixed with 15% cationic liposomes
before inoculation.TheHIV-I
DNA vacclne was amixtureofpcREV
1:5. An appropriate
DNAand pCMV160111B DNAused at aratioof
amount of cytokine plasmids was mixed i n the same PBS. Instead of theliposomes, in some cases, we used I O p g of cholera toxin (CT; List Biologic Laboratories, Campbell, CA) as adjuvant.
The i.n. route has been described in detail elsewhere (36). Mice wereaneathetized with ethyl ether, and after about20 s, 30 pl of the mixture of
DNA vaccine and other reagents were dropped into
the nasal cavity. The
mice inhaled these preparations simply by breathing. The same procedure
employing the same dose of immunogen was used for repeat imlnunizations on days 7 and 21. Five to seven days after
the last boost, immune
responses were studied.

Sample collection
Fecal extract samples were prepared as described elsewhere ( 2 ) . Briefly.
after 100 mg of fecal pellets were mixed with 1 m l of PBS, samples werespun on a vortex mixer, left to settle for 15 min, respun until all materials
were resuspended, and centrifuged at 12.000 rpm. The supernatants werethen collected and tested. Vaginal fluid samples were obtained by themethod described by Staats et al. ( 5 ) .Vaginal wash samples were collectedby flushing the vaginal cavity with I00 pI of sterile PBS while mice were
under isoHurane anesthesia.Estimationsweremadeby comparingcpm
values of the vaginal wash samples of preimmunized and immunized mice.
Each vaginal sample was centrifuged in a microcentrifuge to remove particulate matter. All samples were stored at -20°C until the Ab assay. This
procedure resulted in an approximate IO-fold dilution o f vaglnal samples,as determined by the group referred to above ( 5 ) . and we were able to

Downloaded from www.jimmunol.org on March 10, 2010

DNA encoding the ent' region of the HIV-I 111, strain was exciaed fromclone pSMTE7 (29) by cleavage with Sal1 and Bpull021 and cloned into
the expression plasmid pBCI2KMV at the Hir~dlllandBamHlsitesto
produce pCMV160111B. as reported previously ( I , 30). The pCMV160111B
plasmid, therefore, expresses the precursor gpl60 envelope region of the
HIV-Iprovirusunder
the control of the immediateearlypromotorofCMV. pcREV containa the full-length HIV-I regulatory rei' gene linked to
the CMV promoter sequence end has been shownto efficiently express
HIV-I Rev (31).
Mouse IL- I2 expression plasmid, designated pCAGGSIL- 12, was constructed as follows. Both mouse IL-I2 p35 and p40 cDNAs, kindly provided by Dr. S. Wolf, Genetic Institute, Cambridge, MA, were inserted into
the EwRI site of thepCAGGSexpressionvector(32). resulting i n
pCAGGS-p35 and pCAGGS-p40,respectively.Theexpression
unit forIL-I2 p35. including the CMV immediate early enhancer-chicken p-actin
hybrid promoter. 1L-I2 p35 cDNA, and a rabbit P-globin poly(A) signal,
was excised from pCAGGS-p35 and was inserted downstream of the
1L- 12p40 expression unit of pCAGCS-p40. The pCAGGSIL-12 is described in
Figure I A . The expression plasmid of mouse IL-4, which was a conjugateof the pCAGCS expression vector (Fig.
15).The expression plasmid ofmouse GM-CSF (28. 3 3 ) was provided by Dr. H. L. Davis, Loeb Medical
Research Institute, Ottawa, Canada.

Hindlll3640

INTRANASAL IMMUNIZATION OF D N A VACCINE

confirm this. In this study, we described the value of Ab titers of undiluted
original samples.

Antifusion assay
A previously described antifusion assay was used for measuring the neutralizing activity of the fecal solution (2). CEM cells were infected with
HIV-111, and then cultured for approximately 14 days. Stably infected
CEM cells were first incubated for 2 h with several concentrations of immune sera or with fecal extracts. Uninfected MOLT-I cells were thenadded to this culture system (infected cel1s:uninfected cells = I : 10). Afterthe cells were incubated for 24 h at 3 7 T , giant cells were counted. The
fusion assay evaluation was done in a blinded fashion. The neutralizing
activity was expressed as the percentage of the reduction in syncytium
formation by the fecal extract solution from immunized mice comparedwith that from preimmunized mice. The sameexperiments were performed
several times, and we have provided representative data only.

01
0

1

t

t

Two million regional lymphoid cells and two million spleen cells were
vaccinated and restimulated in vitro in the presence of the same number ofirradiated (30 Cy) syngeneic spleen cells pulsed with V3 (HIV-IIIIB, RG
PGRAFVTI) (2, 5) peptide in a 24-well plate. In other groups, 5 pg/ml of
Con A or 1 pM of ionomycin and 10 ng/ml of PMA were added to theculture systems to examine cytokine synthesis. After 24h, IL-4 inthe culture supernatants was quantitated by an IL-4-specific ELISA. For the IL-4
ELISA, supernatants were collected and mAb BVD6-101 I (anti-IL-4,PharMingen, San Diego, CA) was used as the capturing agent. Then, BVD624G2-biotin (PharMingen) was used as biotinylated developing Ab. For the
IFN-y ELISA, supernatants of the 48-h cultures were collected and mAb
R4-6A2 (anti-IFN-y) was used as the capturing agent. XMG 1.2-biotin(PharMingen) was also used. Avidin-conjugated alkaline phosphatase wasused in a colorimetric assay (39) to detect the presence of bound second Ab
and human cytokine. The concentration of lymphokines was determinedusing a standard curve prepared for recombinant murine IFN-y or IL-4
(both from PharMingen).

Footpad swelling response
The footpad swelling response was measured as previously described (40).
The appropriate amount of DNA vaccine with or without liposomes wasadministered i.n. to mice on days 0, 7, and 21. As a control, we immunizedwith 5 pg of sperm whale myoglobin. For the first 15 days, 300 pg of
purified IgG fraction of sheep anti-mouse IL-12 Ab or 100 pg of XMGl.2(a rat IgGl mAb to mouse IFN-yprovided by Dr. J. Miller DNAX researchInstitute, Palo Alto, CA) was i.v. injected every 2 days. The amount of Ab
injected into mice was almost the same as other previous reports (41-43).
Sixdays after the last vaccination, 25 kg of 111, V3 peptide RIQRG
PGRAFVTIGK (44, 4 3 , 10 pg of gp160 or 2.5 p g of sperm whale myoglobin peptide (ISEAIIHVLHSRHP, Gorai et al. (46)) were injected into
mouse footpads. We also used V3 peptide in the tests of cell-mediatedimmune responses, because the sequence of V3 peptide (RGPGRAFVTI)
was reported to overlap for the CTL and helper cell epitopes of HIV- 1 IIIB
(44, 45). After 24 h, the extent of footpad swelling was estimated as thedifference in thickness in units of IO-* mm between the preinjected andpostinjected footpads.

HIV-specific cytotoxic test
Two micrograms of DNA vaccine with various adjuvants or liposomes
were administered i.n. or i.m. to mice on days 0, 7, and 21. As above, forthe first 15 days 300 pg of anti-lL-I2 Ab or 100 pg of anti-IFN-y Ab wasi.v. injected every 2 days.
Five to seven days after the last DNA vaccination, spleen and regional
immune lymph node cells were collected. Then, 1 X lo6 lymphoid cells
from the immunized mice were restimulated in vitro in the presence of

4

5

6

15

I

t

FIGURE 2. Time course of anti-gpl60 IgC titers in immunized mice.
Two micrograms of DNA vaccine were administered i.n. or i.m. ondays 0, 7, and 21, and serum Ab titers were assayed by gpl60-coated
tiELISA. Arrows indicate the day of immunization. Data are meanters ? SE of five to eight independent mice. Thisis a representative result of three separate experiments. *, Signifies a statistically significant
difference ( p < 0.05) between the group i.m. administered DNA plus
cationic liposomes (A)and i.n. administered DNA plus liposomes (0).

irradiated (30 Cy) syngeneic spleen cells pulsed with 5 pg/ml of V3 peptide (RGPGRAFVTI) (2, 5). After culturing for 5 days, the cytotoxic activity of these spleen cells was measured by a 6-h "Cr release assay using
V3 peptide-pulsed target cells (47). The target cells were prepared using
the same 111, V3 peptide pulsed onto Con A blasts or P815 (H-2d) cells.
These treated cells were labeled with ["'Cr]Na,O, (sodium chromate; Amersham Japan, Tokyo, Japan), washed thoroughly. and used as targets. In
some cases,effector cells were incubated with purified anti-CD8 mAb (Accurate, Westbury, NY) for 1 h at 4°C. After washing, cells were incubatedwith a 1:10 dilution of low-toxicity rabbit complement (Accurate) for 1 h
at 37°C. Cells were then washed and used for the cytotoxicity assay. E:T
ratios ranged from 5: 1 to 80:l. The percentage of specific 5'Cr release wascalculated as 100 X (experimental release - spontaneous release)/(maximun release - spontaneous release). Target cells incubated in medium
alone and with 5% Triton X-I00 were used to determine the spontaneousand the maximum release of chromium, respectively.

Data analysis
All values were expressed as means 2 SE. Statistical analysis of the experimental data and controls was performed using the two-tailed Student's t test.

Results
Serum Ab responses to i.n. immunization
T h e HIV DNA vaccine was administered i.n.on days 0 , 7 , and 2 1,
andantiseracollected
5 daysafterthethirdimmunization.We
initially studied the dose-response curve induced by i.n.DNA vaccination. When we used V3IIIB long peptide as coatingAg for the
ELISA assay, vaccine doses of 0.1, 2, 5 , and I O pg led to meanlog, Ab titers of 2.5 I
1.2 (SE), 8.2 -t 1.8, 9.3 2 2.1, and 10.0 t1.6, respectively. But when we used gp160 asa coating Ag, Ab
titers of sera obtained by 0.1, 2, 5 , and 10 p g of vaccine were
3.6 2 1.4, 9.0 ? 2.4, 10.1 2 2.4,and 11.3 2 2.5,respectively.
Therefore, we generally useda dose of 2 p g of vaccine, and serum
Ab titers were determined usinggp160 as a coating Ag. The serum
Ab titers were compared for mice immunized with 2 /.~gof DNA
injected into three kinds of muscles. The mean Ab titers of sera
obtained by immunization into the gastrocnemius, quadricep, andbicep muscles were 8.7 2 2.0, 8.6 f 1.7, and 8.8 -C I .6, respectively.
From these results and considering the previous reports(33), we used
DNA vaccinaboth quadriceps and gastrocnemius muscles for i.m.
tion. W e next studied the time course over which serum
Abs were
induced following i.n.or i.m. immunization (Fig.2). Two micrograms

Downloaded from www.jimmunol.org on March 10, 2010

Cytokine assays

3

weeks

Neutralization assay using p24 protein detection method
The p24 protein assay was performed by a method described elsewhere (2).
A 4% solution of immune antisera or fecal extracts, as well as columnadsorbed fecal samples from each immunized or preimmunized mouse,
was added to medium containing HIV-I and incubated for 1 h as previously described (2). Uninfected CEM were then added to the medium
containing the virus. After 4 h, the cells were washed and cultured for 5
days. Theconcentrations of p24 protein in the filtered cell-free supernatants
were measured using an HIV-I-specific enzyme immunoassay (Abbott
Laboratories, North Chicago, IL) according to the manufacturer's instructions. The concentration of p24 (ng/ml) was determined from a standard
curve derived from dilutions of the known standard.

2

The Journal of Immunology

3 641

Reciprocal logz antibody titers

Reciprocal lo& antibody titers

151413121110 9

8

7

6

5

4

3

2

1

0

1

1

1

1

1

1

1

1

1

l

1

1

1

1

1

1

0
”_

1 2 3 45

6 7 8 9 1 0 1 1 12 131415

I

I

I

I

I

I

I

I

1

A +

I

I

I

I

I

I

A’

serum
A-“”I-

m IgG
0

E
z
l IgM
IgE

I n t r a m u s c u l a r routeI n t r a n a s a l routeof DNA vaccine, with or without cationic liposomes, were used to
Changes in Ab levels using cytokine expression plasmids or
immunize mice, and Ab titers of each group of animals were comvarious adjuvantspared. Intranasal administrationof the vaccine combined withcationic
The next experiments evaluated adjuvants and cytokines for theirliposomes inducedthe strongest and longest-lasting Ab response (Fig. potential to enhance the production of HIV-specific serum Abs
2). The i.n. route of immunization induced higher titers of HIV-spefollowing i.n. vaccination. As shown in Figure 4, cationic lipocific IgG Abs than did the i.m. route. Moreover, serum IgG Ab titers
somes enhanced serum Ab levels when used in combination withafter 10 mo by i.n. and i.m. immunization were 9.6 t 1.1 and 7.3 t
the vaccine. When the IL-4 or GM-CSF expression plasmid was
0.9, respectively.
included in the vaccine, a significant increase in serum Ab levels
occurred. A CT exhibited no adjuvant activity under this condition.
Coinjection of vaccine plus an IL-12 expressing plasmid also
Comparison of Ig isotype of serum and mucosal Abs
failed to modify HIV-specific serum Ab titers. Coadministered
GM-CSF and IL-12 expression plasmid resulted in Ab titers comThe Ig isotype and titers raised by DNA vaccination of serum,
parable to that of mice given GM-CSF plasmid as adjuvant.
fecalextracts,andvaginal
fluid werethen examined. In our
We then analyzed fecal IgA levels and the immunomodulatory
experiments, the Ab titer of fecal extracts and vaginal fluid byeffectsof adjuvants and cytokine plasmids following i n . adminELISA was clearlydetectedusingthelong
V3 peptideas a
istrationof the vaccine (Fig. 5). Fecal IgA Ab titers were signifcoating Ag (2). We therefore used this peptide in the mucosal
icantly greater after i.n. vs i.m. immunization in mice immunized
Ab assay. gp160 protein was used when the levels of serum Abwith vaccine alone in the absence or presence of liposome. The
were determined. The serum IgG titer was the highest, whereas
coadministration of IL-4 or GM-CSF expression plasmid further
IgM and IgA serum levelswere considerably lower (Fig. 3). IgEincreased the level of fecal IgA. Coadministration of IL-12 exAbs were detectable in serum with i n . immunization only. Fepression plasmids did not modify fecal IgA Ab levels. Again, howcal extract samples contained more IgA than IgG following i.n.
ever, a strong level of anti-V3 Ab was observed when we coadand i.m. vaccination. However, IgA Ablevels were greater thanministered IL-12 plus GM-CSF expression plasmids. In the
IgG levels in vaginal fluids. The titer of mucosal IgA Ab wasabsence of cytokines, fecal Ab titers of i.n. vaccinated groups wererather enhanced when DNA vaccine was i.n. administered. The
significantly higher than the i.m. vaccinated groups. As mentioned
titers of anti-SC Ab in fecal and vaginal samples were 5.3 t 1.2
above, these mucosal IgA patterns persisted for 15 wk.
and 5.5 i 0, respectively, whereas the level of this Ab in serum
Antifusion activity
was undetectable. Compared with the i.m. route, i.n. vaccination elicitedhigherlevelsof mucosal IgA.Seventeen weeks
The capacity of immune antisera to inhibit HIV-mediated fusion
after the last i.n. administration of vaccine, the mean IgA Ab
was then examined. Two groups of 6 to 7 mice were immunized
titers of serum, fecal, andvaginal samples were 4.0 ? 0.6,
i.n. or i.m. (gastrocnemius muscles) three times with the DNA
8.8 t 1.7, and 4.6 ? 0.6, respectively, whereas those obtained
vaccine. The antifusion activity of the resulting antisera were comby i.m. DNA vaccination were 3.5 t 0.2, 5.3 t 1.1, and 3.6 ?
pared. The maximum dilution that inhibited giant cell formation by
0.8, respectively. These results suggest that i n . administration>50% was taken as the antifusion titer. The mean antifusion titerof the DNA vaccine induced higher levels of secretory IgA Ab
of the i.n. DNA administered group was 4.2 ? 0.7, whereas that ofin the mucosas of both gut and vagina than i.m. administration.
the i.m. group was 4.0 i 1.2. The mean titer of the control sera

Downloaded from www.jimmunol.org on March 10, 2010

FIGURE 3. Comparison of the Ig isotypes and titers of serum, fecal extract, and vaginal fluid samples from mice immunized with D N A vaccine
plus liposomes into gastrocnemius muscles or through the i.n. route. O n days 0, 7, and 28, the vaccine was administered. Five days after the third
immunization, fecal extract and vaginal fluidsamples were collected, and Ig isotypes were measured by ELISA using long V3 peptides as a coating
Ag. The data represent the mean ? SE of four tosix mice. All experiments were repeated three times. There exists a statistically significantdifference
between A vs A’, B vs B’, C vs C’, D vs D’, and E vs E’ at p < 0.05.

3642

INTRANASAL IMMUNIZATION OF D N A VACCINE

IX:

0 1.m. route il

7

Id

14

16

i
;

14t

lipo
I+)

- ' no
Ag

lip0

lip0

I-)

(+I

CT

lip0

lip0

IL-4

IL-12 GM-CSF GM-CSF

+

+

lip0

+

IL12

We next determined whether these high-titered antisera or fecal
extracts inhibited the growth of HIV- I. HIV-111, was incubatedwith Ab preparations for 2 h. Virus replication was assayed by thep24 protein binding method. We then compared the inhibition ofp24 production using sera or fecal extracts obtained from i.n. or
i.m. DNA vaccinated mice (Table I). Both groups showed a substantial level of p24 inhibition. The mean inhibition value of preimmune fecal extracts was almost the same as those of control
samples in Table I. There was no statistically significant difference
between the i.n. and i.m. groups. Fecal Abof animals given the
combination of DNA vaccine with IL-4 expression plasmids
slightly inhibited p24 protein production. This inhibition disappeared by adsorption of samples on both IgA and V3 peptide binding columns; however, the inhibition by Abs was weak. Similarly,
coadministration of IL-12 and liposome with the i.m. vaccine did
not modify the fecal response.

lipo

+

lipo

+

GM-CSFGM-CSF+

IL- 12

Table I . HIV-I neutralizing activity of Ab from immunized miceusing a p24 protein assay"
p24 Protein Production(ng/ml)Serum or Fecal Samples

lnhibition of p24 protein production by Ab from immunized
mice+

FIGURE 5. Changes in fecal IgA Ab titersusingvariousadjuvantsand cytokine expression plasmids. Samples werecollected on day 28,
and fecal IgA Abs were assayed. Weused long V3 peptide as a coating
Ag.Data representmeantiters 2 SE of sixto eightmice. Thisis arepresentative result of four separate experiments. Data analysis are
the same as in Figure 4.

+ +from the preimmunized group was 0.8 IC_ 1.2. These results suggestthat the antisera obtained by i.n. or i.m. DNA vaccination wereequally capable of inhibiting HIV-1 -specific fusion.
The antifusion activity of immune fecal extracts was also studied. Using groups of five mice each, the mean antifusion titers of
fecal extracts of mice immunized i.n. or i.m. with the DNA vaccine
were 4.3 IC_ 1.8 and 3.4 5 1.0, respectively. The antifusion titer -C
SE of the preimmunized control group was 1.I 5 1.3. These results again suggested that there was no significant difference in theantifusion activity of i.n. and i.m. immunized animals, although thetiters of antifusion activity were generally low.

lipo

I. i.n.administration
Serum sample15.0
IIIB/REV
Fecal sample
15.3
IIIB/REVIIIWREV + IL-4
liposomes
IIIB/REV
IL-4
liposomes(adsorbed on a HIV-1 V3 peptide

+

+

+

HIV-1 Ills (mean 5 SE)

i 0.8+
2 0.7*
14.6 2 1.7"
25.7 2 3.5

Ag column)

IIIB/REV + IL-4 + liposomes(adsorbed on an anti-lgA column)
11. i.m. administration
Serum sample IllBiREV
Fecal sample IIIB/REV
IIIWREV + IL-12 + liposomes
111. Nonimmunecontrol
Serum sample
Fecal sample

27.9 -C 2.2
17.3 2 1.1+
16.2 i- 2.0*
16.3 5 3.1'

28.3 2 2.9+
29.8 ? s.2*

a Five microliters of fecal
extracts from five to seven BALB/c mice immunized
i.n. or i.m. withD N A vaccine or D N A vaccine plus IL-4 expression plasmid. The
same inclination was observed in three other Independent experiments. * and +show statistically significant differences (*p < 0.05; +, < 0.05) against nonimmune control groups of serum (t)and fecal P ) samples.

CMI responses

The next study examined whether i.n. administration of vaccine
stimulated an enhanced CMI response. The DTH levels of the i.n.
immunized groups were about the same as those of the i.m. immunized groups (Table 11). Administration of IL-12 expression
plasmid enhanced the degree ofHIV
env-specific swelling,whereas coinjection with IL-4 expression plasmid suppressed it.
When IL-12 and GM-CSF expression plasmids were coadministered, the DTH response was greater than that with GM-CSF andcomparable to that with IL- 12. Injection of anti-IL- 12 Ab strongly
inhibited the DTH response. Neither plasmid vector alone (without

Downloaded from www.jimmunol.org on March 10, 2010

FIGURE 4. Changes in serum Ab response titers using various adjuvants and cytokine expression plasmids. On days0, 7, and 2 1 , 2 Kg of
DNA vaccine were administered with various adjuvants (liposomes(lipo), 10 pg of CT, and various cytokine expression plasmids(2 pg of
IL-4,IL-12, and/or GM-CSF). On day 28, each serum was collected,
and Ab titers were measured. Data are mean titers i- SE of seven tonine mice. Identical experiments were performed on two other groupsof mice and produced almost the same results. " and +,Signify statistically significant differences ( p < 0.05) between the marked groupand the D N A vaccine plus liposomes group; signifies a statisticallysignificant difference ( p < 0.05) between two adjacent groups.

IL-4
lip0

IL-12

lip0

+
+

CT

3643

The Journal of Immunology
Table II. footpad swelling response of mice immunized by i.n. administration of HIV-7 DNA vaccine”
Swelling Response (Mean 5 SE X 10”
Immunogens (@nouse)

I . i.n.administration

0.2

pCMVl60111B/REV

0.2 + IL-12
0.2 + IL-12 + anti-IL-12 Ab

lll,-V3

peptide1 4.3 2 1.7*
1 8.9 ? 1.5*

2.0
20.0

6.9 2 2.0**,+9.0 2 0.8’
5.8 -C 1.9
9.3 -C 3.1*
4.4 5 1.6
3.6 5 2.3

0.2.~

1.3.9. 2 0.7f.

20.0

17.3 2 1.8§
15.9 2 2.0
18.6 2 3.6’
14.2 ? 1.2
15.7 ? 2.3

10.0
10.0

2.0 F 0.3

0.2 + IL-4
0.2 + GM-CSF
0.2 + GM-CSF + IL-12mm)

Myoglobin peptide

NT
NT
NT
NT
NT
NT

1.6 -t 1.2
1.9 _t 1.0

II. i.m. administrationpCMVl60111B/REV

0.2 + IL-12
0.2 + GM-CSF
0.2 + IL-12 + CM-CSF
2.0

111. Control

i.n. pCMV empty

i.n. myoglobin

1.7 z 0.1

NT
NT

NT
1.2 -C 0.7
1.5 2 0.7NT

1.4 2 0.5
14.0 F 2.2*

insert DNA) nor sperm whale myoglobin-immunized mice exhibited a swelling response when IIIB peptide was injected.
Experiments were performed to determine whether i n . administration of the DNA vaccine induced a CTL response. BALBk
mice were treated with cationic liposomes and DNA vaccine together with cytokine plasmids. After the last immunization, spleencells were collected and cocultured with V3 peptide-pulsed syngenic spleen cells. As shown in Figure 6A, both i.n. and i.m. immunization with the DNA vaccine alone induced a CTL response.
However, coadministration of vaccine with liposomes, IL-12 plasmid, or GM-CSF and IL-12 expression plasmids induced greater
CTL responses than vaccine alone (Fig. 6B). But GM-CSFexpression plasmids did not show such a strong level of CTL (notshown). In contrast, immunization with liposomes, vaccine, andthe IL-4 expression plasmid resulted in a lower level of CTL induction (Fig. 6B). These CTL responses were significantly suppressed by the in vivo injection of anti-IL-12 or anti-IFN-y Ab(Fig. 6C), suggesting that IFN-y as well as IL-12 play importantroles in these plasmid-mediated immune responses. These results
demonstrate that i.n. administration of DNA vaccine with IL-12
and GM-CSF expression plasmids induced the highest level of
CTL activity of combinations evaluated. Our present results also
demonstrate that i.n. administration of DNA also induced a significant CTL response.
Determination of IgG and subclasses of helper T cells

The Ig subclasses of the i.n. or i.m. immune antisera were determined using ELISA. Figure 7 shows that the mean Ig IgGI/IgG2aratios of antisera obtained by i.n. administration of vaccine andliposomes with IL-4 expression plasmid, of vaccine and liposomeswith IL-12 plasmid, or vaccine and liposomes were 5.1, 0.8, and2.9, respectively. The mean IgGI/IgG2a ratios by i.m. administration of the vaccine with and without liposomes were I .3 and 2.1,
respectively. In addition, when DNA vaccination by the two routes
was compared, i.n. administration induced higher IgCl production
than did the i.m. route, regardless of whether liposomes were employed. The IL-4 expression plasmids help to induce mainly IgCl

Ab, and the 1L-12 expression plasmid helps to increase productionof IgG2a. The coadministration of IL-12 and GM-CSF expression
plasmids enhanced both IgGl and IgG2a Abs.
The last experiment was designed to assay cytokine levels in
culture fluids obtained by 24 or 48 h cocultures of spleen cells
from animals immunized with vaccine and the IL-12 plasmid with
V3 peptide-pulsed syngeneic spleen cells or with nonspecific reagents (Fig. 8). There was no great difference in cytokine production between groups subjected to i.n. or i.m. immunization when
we cultured cells in medium only. However, lymphocytes from i n .
immunized mice showed elevated levels of IL-4 production whenstimulated with Con A or PMA and ionomycin relative to i.m.
immunized mice. In contrast, the level of IFN-y increased in cultures of Con A or PMA and ionomycin to stimulated spleen to cells
from mice i.m. immunized with IL-12 expression plasmid andDNA vaccine. A significant level of IFN-y production was observed in the group when lymphoid cells from i.m. immunized
mice were cultured with V3 peptide-pulsed syngeneic spleen cells.
In contrast, only lymphoid cells from mice immunized i n . with
DNA vaccine plus the TL- I2 expression plasmid expressed significant levels of IL-4 when these cells were cocultured with V3
peptide-pulsed syngeneic spleen cells. Lymphoid cells from nonimmune mice expressed undetectable levels of both IL-4 andIFN-y. These results suggest that i.n. administration of DNA vaccine with the IL- 12 plasmid induces both Thl- and Th2-type
responses.

Discussion
Several studies have shown that i.n. administration of protein Ags
induce strong immune responses (5,8,9,48). However, there havebeen only a few reports of a systematic analysis of i.n. DNA vaccination. The present study shows that i n . administration ofan
HIV-I DNA vaccine induces strong Ab production, particularly
production of mucosal IgA (Fig. 5). We also observed high titers
and persistent serum IgC and fecal IgAAb responses (Fig. 3).

Downloaded from www.jimmunol.org on March 10, 2010

” BALB/c mice were i.n. or i.m. (Muscu/usgastrocnemius)immunized witheach dose of DNAvaccine or myoglobin. Seven days after the second boosting, footpad
swelling responses directed to lll,-V3 peptide (RCPCRAFVTI) were examined. For the first 1.5 days, 0.3 mg of anti-IL-12 Ab (anti-IL-12 Ab) was injected every 2 days.
The data represent the mean 2 SE of four to six mice. All experiments were repeated three times, NT, not tested. * and **, Show a statistically significant difference
against the t and other groups (*, p < 0.05; **, p < 0.01 ). +, Indicates statistically significant differences at p < 0.05 between these two groups. 5, Indicates statisticallysignificant difference beween and other groups.

3644

INTRANASAL IMMUNIZATION OF D N A VACCINE-

M i.n.
Hi.m.

.$ 50
b

control.f! 40

3
0

g 30

rn

s 20
10
0

E/T ratio

mc i.n. (liposomes + IL- 12 + GM-CSF)
70

o-o i.n. (liposomes + IL-121
i.n. (liposomes + 1L-41

A-4

o-o i.n. (liposomes)
a i.n.

50

D-0 control

40

30

20

10

I

f

0
251 1

80 I 1

E/T ratio

70
60

0 IIIB/REV+IL12+liposomes
0 IIIB/REV+liposomes

T

50

m

.e

0

.b
0
9
U

40

E

30

8

20

0

10
0

5: 1

25: 1

80: 1

E/T ratio

FIGURE 6 . The CTL response ofBALB/c mice to V3 peptide-pulsed
target cells. Lymphoid cells from each immunized group were restimdated for 5 days. V3 peptide-pulsed syngeneic spleen cells wereusedas target cells, and restimulated lymphocyteswere used as a source ofCTL. Data are mean titers of four to five independent groups of three
to four mice. Control, lymphoid cells
from nonimmunized mice. A,
Comparison of the CTL response to i.n. vs i.m. vaccination, 8,Adjuvant effect of liposomes or cytokine on the DNA vaccine-mediated
CTL response. C, In vivo inhibition studiesby using anti-cytokine Abs.
For the first 15 days, 0.3 mg of Abs was injected i.p.every 2 days. * and**, Signify statisticallysignificantdifferencesbetween
the i.n. D N A
vaccinated group and other groups (*, p < 0.05; **, p < 0.011.

Downloaded from www.jimmunol.org on March 10, 201060

There have been reports showing that i.n. immunization with influenza hemagglutinin (49), Chlamydia trachontaris (50), Streptococcus mufuns Ag I/I1 (51), or recombinant S. mutans Pac (52)induces Ag-specific serum IgA as well as IgG production. Our
previous studies revealed that oral administration is also effectiveat raising IgA Abs, but that this response was short lasting (2). The
reason for these short periods of high Ab production or oral tolerance (53) is not yet clear; however, turnover of intestinal epithelium is faster than that of upper respiratory or pulmonary tissue.
There is retention of Ag or DNA in the pulmonary tissues. The
pulmonary tissues contain vast areas of epithelial cells that are
directly exposed to the Ag. Since many lymphoid organs lie adjacent to these epithelial cells, this might explain the developmentof a good Ag-specific immune response. However, detailed analyses are necessary to explain the long-lasting high titer Abs induced by i.n. immunization.
HIV-specific IgA Ab titers in both fecal and vaginal fluid werehighest when the vaccine was administered i.n. (Fig. 5), consistent
with other reports (49-52). As shown in Table I, fecal extract
inhibited HIV-I replication, indicating that the IgA was functionally active. But the inhibition by fecal extract was weak. This typeof HIV-I inhibition was also observed using a peptide vaccine as
the immunogen, as has been previously described in detail (2).
Intranasal immunization induced IgA production in vaginal fluid
as well as feces, suggesting this DNA plasmid immunization strategy may be useful in the development of HIV-I vaccines. The
present finding that IgA inhibited HIV-I growth is particularly
important since two of the most common points of entry for thevirus are the anal and vaginal regions. However, it might be stillquestioned whether the method we used for the intramuscular immunization is just the best way or not. We did not use bupivacaine
for preimmune procedures ( 1 1, 12). Thereare several groups using
methods similar to our procedure (DNA in saline). There are several methods of DNA vaccination, such as 50-pg injection into themuscles of mice in PBS or 5 to 20-pg injection by gene gun. We
could not accurately compare these conditions in this study. From
our present data we can at least say that the i.n. route of DNA
plasmid immunization induces high levels of HIV-specificimmunity.
The present study also demonstrates that cationic liposomes significantly enhance immunity when administered i.n. together with
the DNA vaccine (Figs. 2,4,5, and 6). Used as adjuvants, cationicliposomes have been shown to increase the efect of protein immunization (14-16). Systemic eeects with these liposomes havealso been observed with intramuscular DNA vaccination (Okuda etal., manuscript in preparation). Wheeler et al. (10) have reported
that a novel cationic lipid enhances plasmid DNA delivery andexpression in mouse lung. However, to our knowledge, this is thefirst report showing that liposomes enhance immune responses
when coadministered i.n. with a DNA vaccine.
Generally speaking, the i.n. method of administration is usefulfor inducing both IgE and IgA Abs, which are typical indicators ofTh2 cell activation (8, 10, 52, 54). Others have also shown thatmucosal immunization with soluble proteins plus CT as a mucosal
adjuvant induces Th2-like responses (5). The predominance ofTh2-type induction was also observed by our cytokine assay data
(Fig. 8). which showed a rather high IL-4 production and a moderate IFN-y production when i.n. immunization was employed.
The IgGlAgG2a ratio with the i.n. route was higher than with i.m.
administration (Fig. 7), which again suggests an activation of Th2type cells. The characteristics of the strong induction of a Thl-type
response by DNA vaccination (1) was also confirmed by present
studies, and this predominance was activated by the administrationof 1L-12 expression plasmid.

3645

The Journal of Immunology

00.5

I i n . administration

FIGURE 7. Ig subclasses of HIV-specificAb.
On days 0, 7, and 2 1 , 2 p g of D N A vaccine andequal amounts of expression plasmid were administered by the i.n. or i.m. route. After 7 days,
antisera from each group of five to seven mice
were collected, and Ig subclasses were assayedby ELISA using a gpl60 as the coating Ag. Data
represent mean OD ? SE. *, Signifies a statistically significant difference( p < 0.05) between aand other groups. +,Signifies a statistical significant difference( p < 0.05) between band othergroups.

II

O.D. (490nml
1
1.5

2.5

2

, -, , ,IIIB/REV only
IIIB/REV + liposomes

IIIB/REV + llposomes + IL-12
IIIB/REV + liposomes + 1L4
IIIB/REV + liposomes + IL-12+ GM-CSF

I1 i.m. administration

I+*”””””- ””“

I.

IIIB/REV only

3

IIIB/REV + liposomes

IFN-y production

IL-4 production

17 i.n. vaccination

PMA*

Mediumi.m. vaccinationI

non-immune

I

”

f

ionomycinionomycin

IF+

1

P

I

I

I

0

200

400

600

IL-4 (pg/ml)

I

1

i.m. vaccination
non-immuneJf

V3 peptidekE

0 i.n. vaccinationL
V3 peptide

P

I

0

10

F

I

I

I

20

30

40

IFN-y (ng/ml)

FIGURE 8. Influence of the route ofDNA vaccination on cytokine production. Two micrograms each of IIIB/REV and IL-12 expression plasmids
were immunized i.m. or i.n. into mice on days 0, 7, and 21. The immune lymphoid cells were collected and cocultured with each reagent. Meanlevels f SE of cytokines were assayed. Data represent mean cytokine levels2 SE of five to seven mice. These dataare representative of three otherindividual experiments.

We detected more IgE Ab in the sera of mice immunized by thei.n. vs i.m. routes (Fig. 2). IgE production by i.n. or perioral administration has been reported by others ( 3 ,which is a sign ofTh2-type response. It is uncertain at this time whether the serum
IgE Ab seen with i.n. DNA vaccination is sufficiently high to cause
anaphylaxis or allergy. There is a report that anti-DNA Abs induced by DNA vaccination do not cause autoimmune syndromesin any autoimmune model mice (55). Our mice demonstrated noclinical disease. We also used CT as an adjuvant but did not observe any significant increases in Ab titers under our experimental
conditions. Many studies (2, 5 , 8) have shown that CT helps toincrease the immunogenicity of various peptide or protein-containing vaccines. In the future, we might find conditions under whichthese adjuvants would be more effective. IL-12, an important factor in the differentiation of Thl cells (19), has been reported toincrease I ) NK cell and CTL activities (20, 26); 2) T cell proliferation (56); and 3) Thl cell maturation and production of IFN-y(21). In addition, IL-12 inhibits the progression of various intracellular pathogens in mice (22, 23) and protects against the devel-

opment of murine AIDS (24). These reports were relevant to thedevelopment of our DNA vaccine for HIV- I .
We predicted that the Th2-type response might even be suppressed by coadministration of the IL- 12 expression plasmid, since
it has been reported that IL-12 inhibits IgE response (23).The
tendency for IL- 12 expression plasmids to enhance IFN- y production and somewhat suppress IL-4 production was also observed i ncytokine assays (Fig. 8). These observations are very common in
the course of Thl or Th2 maturation.
Our results revealed that coadministration of an IL- I2 expression plasmid with an HIV DNA vaccine did not reduce IgA Ab
production or levels of serum Ig (Figs. 4 and 5). There are several
reports that IL-12 does not suppress Ab production (25, 26). It did
in fact strongly enhance both DTH and CTL responses. This isquite similar to the sequence of events in CTL activation by live
attenuated vaccines, and the intracellular steps involved are essential for inducing a strong CTL production (25). Our present study
demonstrated that administration of IL- 12 expression plasmid induces IFN-y production (Fig. 8), which also plays an important

Downloaded from www.jimmunol.org on March 10, 2010

LL

Medium3646

Acknowledgments
We thank T. Kaneko and A. Honsho for technical assistance, and S. Wolf.
Genetic Institute. Boston, MA, for providing the IL-12 expression plasmid.
We ala0 thank H. L. Davis, Loeb Medical Research Institute, Ottawa, Canada, as well as D. Klinman, DVP, C B E W D A , Betheada, MD, for useful
discussions and advice on the manuscript.

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Downloaded from www.jimmunol.org on March 10, 2010role in CTL induction, because in vivo injection of anti-IFN-y Ab
strongly suppressed CTL responses (Fig. 6C). The results of cytokine assays (Fig. 8) and Ig subclass determinations (Fig. 7) also
revealed that the coadministration of DNA vaccine with 1L- 12expression plasmid preferentially activated Thl -type cells. In anotherset of experiments, we injected mice i.v. with the IL-12 protein toconsider whether this protein was able to increase DTH or CTL
responses. The Th I-type responses were weakin these studies
compared with the present coinjection of IL- 12 expression plasmid(Okuda et al., manuscript in preparation). One explanation for thisis that protein decay in the mouse is fast, and the effect of IL- I2 isonly transient (20); therefore, the continuous production of or stimulation by the IL-12 expression plasmid might be important for
activation of Thl-type cells in this animal (20). In addition, coadministration of GM-CSF also activated CMI. This is the first observation that this type of strong enhancement was induced bycoadministration of both GM-CSF and the 1L-12 plasmid. This
coactivation of GM-CSF and IL-12 expression plasmids isvery
important for not only the HIV-1 vaccine but also for many othervaccine. The activation of both Thl and Th2 is one of the ideal
adjuvant. The mechanism of this type of coactivation is still unclear. However, we conjecture that GM-CSF activates or helps togrow APC, that IL-I2 activates ThO to Thl maturation, and thatThl and Th2 do not interfere with each other.
Our present study clearly demonstrates that DNA vaccine with
anIL- 12 expression plasmid induces good levels of both HIVspecific mucosal IgA Ab and CTL activity. Good enhanced HIVspecific immunities were also observed by coadministration of
IL- 12 and GM-CSF expression plasmids. Intranasal administrationof DNA preparations appears to be an effective method for delivery, because it is simple, easy, and safe, and involves no infectious
microbes along with their associated side effects.

INTRANASAL IMMUNIZATION OF DNA VACCINEThe journal of Immunology

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