1. Published Ahead of Print 6 May 2009.
2009, 83(14):7166. DOI: 10.1128/JVI.00374-09.J. Virol.
Nabel
Schwartz, C. Richter King, Jason G. D. Gall and Gary J.
Kong, Masaru Kanekiyo, David Einfeld, Richard M.
Lingshu Wang, Cheng Cheng, Sung-Youl Ko, Wing-Pui
Enhanced Mucosal Cellular Immunity
Vaccine Vectors to the Intestine Induces
Delivery of Human Immunodeficiency Virus
http://jvi.asm.org/content/83/14/7166
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3. of rAd vectors through the p.o. route and develop effective
delivery of rAd5 and rare serotype rAd35 vectors as gut mu-
cosal HIV vaccines, we have analyzed the obstacles to p.o.
immunization, characterized vector transgene expression, and
systematically compared immune responses induced by p.o.
and local immunization strategies. These studies demonstrated
that the higher immune responses were strongly associated
with higher gene expression in the intestine and support fur-
ther study of gut mucosal immunization in SIV challenge mod-
els as a potential HIV vaccine strategy.
MATERIALS AND METHODS
Animals and viruses. Six- to 8-week-old female BALB/c mice were purchased
from Jackson Laboratories and housed in the experimental animal facility of the
Vaccine Research Center, NIAID, NIH (Bethesda, MD). All animal experi-
ments were reviewed and approved by the Animal Care and Use Committee,
VRC, NIAID, NIH, and performed in accordance with all relevant federal and
NIH guidelines and regulations.
rAd5 vectors are replication-defective E1-, E3-, and E4-deleted human Ads,
and rAd35 vectors are E1- and E3-deleted replication-defective vectors. rAd35/
41L and rAd35/41S vectors are rAd35-based vectors with chimeric Ad41 long
fiber or short fiber, respectively. rAd5-luc, rAd35-luc, rAd35/41L-luc, and rAd35/
41S-luc encode luciferase reporter genes under the control of the cytomegalo-
virus promoter/enhancer. rAd5-gp140B, rAd35-gp140B, and rAd35/41L- or
rAd35/41S-gp140B (rAd35 with Ad41 long or short fiber, respectively) encode
gp140⌬CFI⌬V1V2 of HIV-1 clade B (20). Ad vectors were propagated in 293-
ORF6 cells and purified by cesium chloride gradients (6).
Mouse intestinal explants. Explant culture medium consisted of Dulbecco’s
modified Eagle’s medium–F-12 medium supplemented with 5% fetal bovine
serum, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 g/ml),
gentamicin (50 g/ml), and epidermal growth factor (10 U/ml). All media and
supplements were purchased from Invitrogen, Inc. (Carlsbad, CA). Mouse in-
testines were isolated and cut open longitudinally. After being washed thor-
oughly, the tissues were cut into 5- by 5-mm pieces and placed on presoaked
Gelfoam (Pfizer, New York, NY), with the epithelium uppermost, in 24-well
plates containing 0.3 ml of culture medium. Four microliters of virus solution
containing 1 ϫ 108
to 5 ϫ 108
virus particles (VP) of treated or untreated rAd
vectors encoding luciferase was applied directly to the upper surface of each
explant. After overnight incubation, the tissues were rinsed in phosphate-buff-
ered saline (PBS) and placed in 0.1% Triton X-100. Following a 1-min ultra-
sound treatment and three freeze-thaw cycles, the lysates were centrifuged at
2,000 rpm for 10 min. The supernatants were used for a luciferase assay. The
luminescence was measured using a microplate scintillation and luminescence
counter (PerkinElmer, Shelton, CT). The protein concentration of the cleared
supernatant was determined using a DC protein microplate assay kit (Bio-Rad,
Hercules, CA). The results are shown as relative luminescence units per g
protein.
For the acid and protease sensitivity study, rAd5-luc was (i) treated with 0.1 M
HCl and 2.5 mg/ml pepsin from porcine gastric mucosa (Sigma, St. Louis, MO)
for 5 min at 37°C and neutralized with 10ϫ THB (200 mM Tris, 500 mM HEPES,
1.5 M NaCl), (ii) treated with 25 g/ml trypsin from bovine pancreas (Sigma, St.
Louis, MO) or 2.5 U/ml chymotrypsin (Sigma, St. Louis, MO) for 30 min at 37°C,
or (iii) exposed to the combination of treatments with HCl-pepsin and trypsin
and/or chymotrypsin and then applied to the tissue explants.
To test the susceptibility of intestinal segments to Ad vectors, mouse duode-
num (1 cm downstream of the stomach), jejunum (median part of the small
intestine), ileum (from the junction of the ileum and cecum to 2 cm upstream of
the cecum), and colon (2 cm downstream of the cecum) were isolated and either
washed thoroughly (native) or gently stripped to remove the mucus for culture.
Ten pieces from three mice for each segment were cultured with 4 l virus
solution containing 1 ϫ 108
VP of rAd5-luc and harvested as described above.
Treatment of intestinal explants with DTT, hyaluronidase, and exoglycosi-
dases. The mouse intestinal explants were either not treated or treated with
dithiothreitol (DTT) (20 mM, catalogue number 43816; Sigma, St. Louis, MO) or
hyaluronidase (1 or 10 mg/ml, catalogue number H 3506; Sigma, St. Louis, MO)
for 30 min or exoglycosidases from Trimeresurus cornutus (1, 5, or 10 mg/ml;
Seikagaku Biobusiness, Tokyo, Japan) for 2 min at 37°C. The explants were then
rinsed in the prewarmed medium and cultured with 4 l virus solution containing
1 ϫ 108
VP of rAd5-luc. The stripped jejunum and the untreated ileum were
used as the positive controls, while the medium only was used for the negative
controls. The tissue explants were harvested as described above.
Gene expression in mouse intestine transduced with rAd5-luc in vivo. Three
BALB/c mice per group were either administered p.o. 1010
VP of rAd5-luc in 500
l PBS or given an intraileal injection with 1010
VP of rAd5-luc in 100 l PBS.
Twenty-four hours after injection, the mice were sacrificed, and intestinal seg-
ments, including duodenum, jejunum, ileum, colon, and mesenteric lymph nodes
(MLN), were lysed and assayed for luciferase activity. The group injected with
100 l rAd5 empty vector via the ileum lumen was used as the control. The
intraileal-injection procedure was described previously (17).
Immunization. For the immunogenicity study, immune responses induced by
a single injection of rAd5 encoding HIV-1 gp140B into the ileum lumen were
tested. Five mice from each group were immunized with 1010
VP of rAd5-gp140B
vector in 500 l PBS p.o. or with 1010
VP of rAd5-gp140B vector in 100 l PBS
via ileal injection as described above. Mice that received rAd5 empty vector p.o.
or via ileal injections were used as the negative controls. Three weeks after
inoculation, peripheral blood mononuclear cells (PBMC), spleens, and small
intestines were collected for H-2Dd
/PA-9 tetramer staining. The spleens were
also used for detecting HIV-1 peptide-specific cytokine-producing CD4ϩ
or
CD8ϩ
T lymphocytes. The serum immunoglobulin G (IgG) antibodies were
determined by enzyme-linked immunosorbent assay (ELISA).
In the prime-boost experiments, five mice per group were primed with 1010
VP
of rAd5-gp140B via p.o. dosing or with rAd5, rAd35, Ad35/41L, and rAd35/41S,
all encoding gp140B, via intraileal injections. Three weeks after immunization,
blood was collected and PBMC were isolated and stained for H-2Dd
/PA-9
tetramer. These mice were then boosted intramuscularly (i.m.) with rAd5-
gp140B at 109
VP. Two weeks after the boost, PBMC, spleens, MLN, and small
intestines were harvested for H-2Dd
/PA-9 tetramer staining, and the spleens
were also used for detecting HIV-1 peptide-specific cytokine-producing CD4ϩ
or
CD8ϩ
T lymphocytes. The IgG and IgA antibodies in the sera and vaginal washes
were determined by ELISA.
Lymphocyte isolation. The isolation of lymphocytes from blood, spleen, and
MLN was described previously (17). For lymphocyte isolation from the gut, the
small intestines from individual mice were collected in cold medium. The intes-
tine was opened and washed thoroughly following excision of Peyer’s patches.
Intestinal sections were then minced and digested with 0.5 mg/ml collagenase II
(C-6885; Sigma) for 30 min at 37°C. The resulting supernatants were filtered
through a 40-m cell strainer. After centrifugation, the cell pellets were sus-
pended in 6 ml of 40% Percoll (Amersham Biosciences, Piscataway, NJ) and
overlaid on 4 ml of 75% Percoll. The samples were then subjected to centrifu-
gation at 800 ϫ g for 20 min at 22°C. The cells from the interface of 75% and
40% Percoll were collected, washed with a large volume of medium, and
counted.
Tetramer staining and ICS. Lymphocytes isolated from blood, spleen, MLN,
and the small intestine were used for tetramer staining. The splenic lymphocytes
from individual mice were used for intracellular cytokine staining (ICS), while
the combined intestinal cells from the group of five mice were also used for ICS
in the rAd35/41 chimeric fiber experiment, using ViViD dye (Invitrogen Life
Sciences, Carlsbad, CA) to exclude nonviable cells. The detailed methods for
tetramer staining and ICS were described previously (M. Honda, R. Wang, W.-P.
Kong, M. Kanekiyo, W. Akahata, L. Xu, K. Matsuo, K. Natarajan, H. Robinson,
T. E. Asher, D. A. Price, D. C. Douek, D. H. Margulies, and G. J. Nabel,
submitted for publication; 17).
ELISA for IgG and IgA antibodies to HIV Env. Levels of HIV gp140B-specific
IgG and IgA in the sera or vaginal washes of vaccinated mice were assessed using
ELISA (17). The sera were diluted at 1:1,000 for IgG and 1:50 for IgA, while the
vaginal washes were diluted at 1:3 for the detection of both IgG and IgA.
Absorbance at 450 nm was determined by a Spectra Max instrument (Molecular
Devices).
Data and statistical analysis. All results are presented as means with standard
errors. Statistical analyses were performed based upon comparisons between the
control groups and the treated groups or between differently treated groups by
using the two-tailed Student t test. P values of less than 0.05 were considered
statistically significant.
RESULTS
Determination of barriers to effective delivery of rAd5 vec-
tors to the GI tract. To identify potential mechanisms that
inhibit transduction of the GI tract after rAd gene transfer
after p.o. delivery, we first examined the transduction of mouse
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4. intestinal explants by an rAd5 vector expressing luciferase.
Intestinal explants of the duodenum, jejunum, ileum, and co-
lon were cultured ex vivo, and the relative rAd transduction
efficiencies were determined. The ileum and the colon were
transduced at significantly higher levels than were the duode-
num and jejunum (Fig. 1A, open bars). To determine whether
the superficial layer played a role in the inhibition of transduc-
tion of intestinal cells by rAd, mucus was removed by mechan-
ical and biochemical means. Mechanical removal of the mucus
significantly enhanced the transduction activity in all segments
(P Ͻ 0.05 for the duodenum and jejunum, P Ͻ 0.001 for the
ileum) except for the colon (Fig. 1A, filled versus open bars).
The differences between transductions of native and mucus-
stripped explants were 100-fold in the duodenum and jejunum
and 10-fold in the ileum. Previous in vitro studies have shown
that intestinal mucus can also be dissolved by a variety of
agents, including enzymes, detergents, and sulfhydryl com-
pounds (1, 28). The effect of DTT, hyaluronidase, and glyco-
sidases on the transduction of intestinal explants by rAd5 was
investigated using explants treated and exposed to vector ex
vivo. Transduction of explants treated with DTT was signifi-
cantly higher than transduction of those that were untreated,
specifically in the upper intestinal segments, duodenum and
jejunum (Fig. 1B, filled versus open bars). When mouse jeju-
num was treated with hyaluronidase or exoglycosidases (see
Materials and Methods), transduction was significantly higher
than that of the jejunum treated with medium (Fig. 1C and D,
1 mg/ml). However, high concentrations of these enzymes did
not enhance transduction, possibly due to nonspecific cell
damage.
We next examined the effect of acid and selected proteases
on rAd5 transduction of mouse intestinal explant cultures.
Gastric acid and pepsin are the major components secreted
into the stomach, and trypsin and chymotrypsin are the major
proteases produced by the pancreas. To mimic the passage of
rAd through the gastric environment and into the proximal
section of the small intestine, rAd5-luciferase vector was
treated with combinations of acid and protease and then ap-
plied to the tissue explants. Although we did not observe a
significant decrease in luciferase activity after acid-pepsin,
trypsin, or chymotrypsin treatment alone, the combination of
acid-pepsin treatment with either trypsin or chymotrypsin
treatment significantly reduced transgene expression (Fig. 2A,
lanes 5 and 6). The combination of acid-pepsin with both
trypsin and chymotrypsin further reduced luciferase activity, to
approximately 100-fold lower than transduction with untreated
rAd5-luc (Fig. 2A, lane 7). This result suggested that rAd5
vectors were susceptible to degradation by gastric proteases.
Taken together, these ex vivo experiments indicated that mul-
tiple barriers reduce transduction of intestinal epithelia by
rAd5 vectors and demonstrated differential transduction of
specific intestinal segments.
Development of intraileal delivery to increase gene delivery
to the small intestine. Since the untreated ileum was trans-
FIG. 1. The distal regions of the intestine were efficiently transduced by rAd5, and removal of luminal substances increased transduction of the
proximal intestine. (A) Efficient transduction of intestinal segments. Segments of intestine were left untreated (native), or the mucus was gently
removed (stripped) and then transduced with rAd5-luc. (B) Treatment of intestinal segments with DTT increased the transduction efficiency of
rAd5-Luc. (C and D) Effect of removal of the glycocalyx and hyaluronic acid on transduction. Mouse jejuna were treated with hyaluronidase (C) or
mixed glycosidases (D) prior to transduction with rAd5-luc. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. RLU, relative luminescence units.
7168 WANG ET AL. J. VIROL.
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5. duced most effectively in ex vivo organ culture, we developed
an in vivo route of administration that targeted the ileum and
bypassed the significant barriers encountered by rAd in the GI
tract. It has been reported that receptor binding and uptake of
rAd5 occur relatively quickly, with the majority (80 to 85%) of
the bound virions being taken up by permissive cells within 5 to
10 min (12). We found that a 10-min exposure of ileum ex-
plants to rAd5 resulted in levels of transgene expression similar
to those obtained with longer time exposures (see Fig. S1 in
the supplemental material). Therefore, for in vivo studies,
the ileum loop was formed to hold the virus solution for 20 min
to ensure efficient binding of viruses within the intestine and
the separation of virus from undigested food in the ileum.
To determine the relative efficiency of rAd5-mediated trans-
duction, mice were administered rAd5 (1010
VP) encoding
luciferase either p.o. or via the intraileal-injection method.
Twenty-four hours later, mice were sacrificed, and the intesti-
nal segments were assayed for luciferase activity. A low level of
luciferase activity was detected in the ileum after p.o. admin-
istration, but the luciferase activity was not significantly differ-
ent from that for the empty rAd5 control group (Fig. 2B) (P Ͼ
0.05). Following intraileal injection, there was approximately a
10- to 100-fold-higher luciferase activity in the ileum and MLN
than for the control group (Fig. 2B) (P Ͻ 0.05). Importantly,
the luciferase activity in the ilea from injected mice was signif-
icantly higher than that for the p.o. dosed mice (Fig. 2B) (P Ͻ
0.05).
Immunization via intraileal injection generated humoral
and cellular responses in both mucosal and systemic compart-
ments. The higher efficiency of intestinal cell transduction ob-
served with ileal injection than with p.o. dosing suggested the
possibility that intraileal administration of rAd vectors would
FIG. 2. Transduction of intestinal explant cultures by rAd5-luc was reduced by exposure to low pH and proteases and enhanced gene expression
by intraileal delivery of rAd5 in vivo. (A) The rAd vector encoding luciferase was treated as indicated (ϩ, treatment; Ϫ, no treatment) and applied
to explants of mouse ileum. *, P Ͻ 0.05. (B) Mice were administered rAd5-luc via p.o. or intraileal injection, and the levels of luciferase activity
in intestinal segments and MLN were determined 24 h later. *, P Ͻ 0.05. RLU, relative luminescence units.
VOL. 83, 2009 INTRAILEAL rAd5 VACCINE STIMULATES MUCOSAL IMMUNITY 7169
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6. induce a more potent immune response to the encoded trans-
gene. Mice received a single administration of rAd5-gp140B
(1010
VP) p.o. or via intraileal injection. Three weeks later, the
mice immunized with rAd5-gp140B via intraileal injection
showed significantly higher levels of H-2Dd
/PA-9ϩ
CD8ϩ
T
cells in both the small intestine and the systemic compartments
than did the mice immunized p.o. (Fig. 3A). Oral administra-
tion did not result in detectable responses higher than back-
ground (Fig. 3A) (P Ͼ 0.05). The average levels of H-2Dd
/
PA-9ϩ
CD8ϩ
T cells in the mice given an intraileal injection
FIG. 3. Mucosal and systemic immune responses induced by a single intraileal injection of rAd5-gp140B. Mice were immunized with
rAd5gp-140B (rAd5) or rAd5 empty vector (Ϫ) via the p.o. or the intraileal route as indicated. (A) Percentages of HIV-specific H-2Dd
/PA-9ϩ
CD8ϩ
T cells from PBMC, spleens, and small intestines. (B) IgG antibody in the serum. OD450nm, absorbance at 450 nm. (C) Percentages of
HIV-specific IL-2-, IFN-␥-, and TNF-␣-secreting CD4ϩ
or CD8ϩ
T cells from the spleens. The data from each individual mouse and the mean
values from the five mice (black bars) are shown. *, P Ͻ 0.05; **, P Ͻ 0.01.
7170 WANG ET AL. J. VIROL.
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7. were 7.11% (ranging from 2 to 11%) in PBMC, 3.47% (ranging
from 1.44 to 5.82%) in the spleen, and 5.53% (ranging from
2.13 to 9.65%) in the small intestine. HIV Env-specific IgG
responses were also detected in sera from mice immunized via
the intraileal injection but not in sera from the p.o. dosed mice
(Fig. 3B). ICS analysis of splenocytes was consistent with the
tetramer staining results; numbers of HIV-1-specific CD4ϩ
and CD8ϩ
T cells producing interleukin-2 (IL-2), gamma in-
terferon (IFN-␥), and tumor necrosis factor alpha (TNF-␣)
were significantly higher in the mice that received rAd5-
gp140B via intraileal injection than in the mice that received
equal amounts of rAd5-gp140B p.o. (Fig. 3C). In this analysis,
the magnitudes of CD8ϩ
T-cell response differed between tet-
ramer staining and ICS, suggesting that there was a dispropor-
tionate representation of the dominant tetramer response or
that not all of the tetramer-positive CD8ϩ
T cells were secret-
ing the measurable cytokines, a dichotomy described to occur
previously when using these techniques (42). Tetramer staining
measures CD8ϩ
T cells responding to an immunodominant
HIV-Env peptide regardless of function, including inactivated
cells and activated cells, while ICS detects only activated cells
or functional cells. Oral immunization generated only a back-
ground level of cytokine-producing CD4ϩ
or CD8ϩ
T cells.
These data demonstrate that the rAd5 vector induced substan-
tial antigen-specific cellular and humoral immune responses
after a single intraileal immunization, while p.o. immunization
of the same amount of rAd5 did not induce any detectable
immune responses.
A mucosal prime-systemic boost regimen elicited potent cel-
lular immunity in the small intestine and the systemic com-
partments. The relative potencies of p.o. and intraileal routes
of administration as primes for systemic boosting were next
examined. Mice were primed on day 0 with empty rAd5 or
rAd5-gp140B p.o. or via intraileal injection and were boosted
i.m. on day 21 with rAd5-gp140B. Before the boosting step,
PBMC were harvested for tetramer staining, and consistent
with the results described above, immunization with rAd5-
gp140B via intraileal injections induced significantly higher
responses than p.o. administration (Fig. 4A). After the mice
were boosted with rAd5-gp140B i.m., the mice primed via the
intraileal route showed a dramatic increase in antigen-specific
H-2Dd
/PA-9ϩ
CD8ϩ
T-cell responses in both systemic and
mucosal compartments compared to the responses of the
group primed p.o. or i.m. (Fig. 4B). HIV-1 peptide-specific
cytokine-producing CD8ϩ
T cells and CD4ϩ
T cells were also
boosted effectively in the mice given an ileal prime and an i.m.
boost. On average, 12.44% of CD8ϩ
splenic T cells produced
IFN-␥, and 10.65% of CD8ϩ
splenic T cells were TNF-␣ pos-
itive (Fig. 4C). The mice primed p.o. or via ileal injection and
boosted i.m. generated higher levels of HIV-1 peptide-specific
IFN-␥- and TNF-␣-producing CD4ϩ
T cells than the mice
immunized by i.m. injection only (Fig. 4C). There were no
significant differences in CD4 frequencies between the prime-
boost groups. The only differences were relative to the single-
immunization group, indicating that prime-boost regimens in-
duce higher frequencies of immune cells than a single
immunization. HIV-1-specific IgG and IgA antibodies were
also detected in sera and vaginal washes, but the differences
between the mice primed p.o. and the mice primed by ileal
injection were not significant (see Fig. S2 in the supplemental
FIG. 4. Immune responses induced by ileum prime and i.m. boost.
Mice were administered rAd5gp-140B (rAd5) or empty vector (Ϫ) via
the p.o. or intraileal route and boosted i.m. (ϩ) 3 weeks later. (A) Per-
centages of HIV-specific H-2Dd
/PA-9ϩ
CD8ϩ
T cells from the PBMC
immediately prior to the i.m. injection of rAd5-gp140B. (B) Percent-
ages of HIV-specific H-2Dd
/PA-9ϩ
CD8ϩ
T cells from the PBMC,
spleens, MLN, and small intestines at 2 weeks after the i.m. injection
of rAd5-gp140B. (C) HIV-1-specific IFN-␥- and TNF-␣-secreting
CD4ϩ
or CD8ϩ
T lymphocytes from the spleens 2 weeks after i.m.
injection of rAd5-gp140B. The data from each individual mouse and
the mean values from the five mice (black bars) are shown. *, P Ͻ 0.05;
**, P Ͻ 0.01; ***, P Ͻ 0.001.
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8. material). These results demonstrate that priming directly in
the ileum and boosting i.m. induced the most potent CD8ϩ
T-cell immunity in the small intestine as well as in the systemic
compartments.
The fiber proteins of Ad41 did not increase the mucosal
immunogenicity of a rare serotype vector, rAd35. The swap-
ping of Ad5 fiber proteins into Ad35 has previously been
shown to increase immunogenicity (29). Since we have seen
intestinal and systemic immune responses induced by rAd41
vectors of a magnitude similar to that of responses induced by
rAd5 (17), we determined whether the swap of Ad41 proteins
into rAd35 would affect rAd35-mediated transduction and im-
mune responses. Explant cultures of murine intestine were
transduced with rAd35, rAd35/41L, and rAd35/41S, encoding
luciferase at a 1- to 2-log-lower efficiency than that for rAd5
(Fig. 5A). We also compared the relative potencies of the
rAd35 and rAd5 vectors in priming for an i.m. rAd5 boost.
While an intraileal prime-i.m. boost with rAd5-gp140B elicited
significantly higher levels of CD8ϩ
T-cell responses in the
spleen and in the small intestine than did rAd5 i.m. alone,
intraileal administration of rAd35, rAd35/41L, and rAd35/41S
failed to increase the rAd5 i.m boost (Fig. 5B and C, right, lane
3 versus lane 2, 4, 5, or 6). However, CD4ϩ
T-cell responses did
not show a difference after rAd5 boost, possibly due to the
lower magnitude of responders than CD8ϩ
cells typically seen
with rAd5 immunization. In addition, an ileal prime and i.m.
boost with rAd5-gp140B induced a twofold-greater IFN-␥ϩ
CD8ϩ
T-cell response in the small intestine than did rAd5 i.m.
alone. In contrast, an ileal prime with rAd35, rAd35/41L, and
rAd35/41S did not show a boost response in the small intestine
higher than that with rAd5 i.m. alone (see Fig. S3 in the
supplemental material). These results demonstrate that the
direct intraileal injection of rAd5, but not that of rAd35,
rAd35/41L, and rAd35/41S, could prime for an i.m. rAd5 boost
to generate potent immune responses in the systemic and mu-
cosal compartments.
DISCUSSION
The intestinal mucosa represents a primary site of HIV
replication during the peak of infection (3, 23, 26, 43). Vacci-
nation through the GI mucosa could potentially induce muco-
sal immunity to prevent virus entry and/or limit the spread of
infection. Oral immunization is the most convenient way to
deliver mucosal vaccines. Although several studies have re-
ported that the use of this route of immunization of Ad vectors
encoding various virus antigens generated antibody responses
in the serum and mucosal secretions (33, 37), the administra-
tion of Ads encoding Gag or Env from HIV-1 or SIV elicited
weak or undetectable intestinal mucosa responses (24, 30). The
major obstacles to successful induction of mucosal immunity
and/or systemic immunity via GI delivery of vaccine have not
been defined previously. In this study utilizing murine intesti-
nal explant cultures and in vivo gene expression assessments,
we demonstrate that the combination of a low-pH environ-
ment and the presence of proteases as well as the mucus/
glycocalyx in the GI tract prevents rAd5 from effectively trans-
ducing the intestinal cells when delivered p.o. Therefore, p.o.
immunization of vaccine vectors must overcome or bypass
these barriers in the GI tract in order to be effective. We also
show that the ileum is the most receptive site for rAd trans-
duction and that intraileal injection of rAd5 results in a signif-
icantly higher transgene expression in the ileum than does p.o.
delivery. In addition, intraileal immunization with rAd5 did not
induce significant systemic antivector neutralizing activity,
since the rAd5 i.m. administration was effective in the homol-
ogous rAd5 prime-rAd5 boost regimen. This is in agreement
with our previous work demonstrating the absence of circulat-
ing neutralizing antibodies following p.o. administration of
rAd5 and rAd41 (17). It has also been shown that preexisting
immunity to the vaccine carrier did not impair p.o. vaccination
with Ad vectors in mice (45). Thus, the ileum represents a valid
target for delivery of rAd vectors.
Utilization of a route of administration that bypassed the
barriers to ileal transduction revealed the potency of rAd5 as a
mucosal immunogen. A single immunization of mice with rAd5
encoding HIV-1 gp140B via intraileal injection generated sig-
nificantly higher numbers of H-2Dd
/PA-9ϩ
CD8ϩ
T cells in
both mucosal and systemic compartments and antigen-specific
IFN-␥-, TNF-␣-, and IL-2-producing CD4ϩ
and CD8ϩ
T cells
in the spleen than did p.o. administration (Fig. 3A and C). In
fact, p.o. immunization did not generate any detectable im-
mune response after a single dose in our study. These results
indicate that the higher transduction from intraileal injections
than from p.o. delivery (Fig. 2B) is associated with the stronger
immune responses to HIV-1 gp140B. Systemic boosting with
rAd5-gp140B dramatically increased CD8ϩ
T-cell responses in
the mice primed via the intraileal route in both systemic and
mucosal compartments compared to responses in the group
primed p.o. (Fig. 4B and C, right) as well as splenic CD4ϩ
T-cell responses compared to those in the mice primed p.o. or
via ileal injection (Fig. 4C, left). Whether the activation of
CD4ϩ
T cells would provide more target cells for HIV acqui-
sition will be addressed in nonhuman primate (NHP) studies,
though a previous study in which such mucosal responses were
detected showed that these responses led to protection (21,
25). Oral dosing with replication-competent Ad vectors gener-
ates systemic immune responses in nonhuman primates, but
multiple primes and protein boosting are required for protec-
tive efficacy against SIVmac251 challenge (32, 47). Oral vacci-
nation with replication-competent Ad4 and Ad7 has been
shown to generate protective Ad neutralizing antibodies in
humans (11), indicating that replication-competent vectors
elicit antivector neutralizing antibodies, which may compete
with transgene-specific responses or may prevent multiple p.o.
administrations of the same vector and affect efficacy. In addi-
tion, the magnitude and contribution of the transgene-specific
response in mediating protection by replication-competent
rAd remain unknown in this model because the gut mucosal
immune responses were not measured and compared to sys-
temic responses. Thus, there are potential efficacy and regula-
tory concerns with replication-competent rAds, and further
studies are needed to assess the potential efficacy of gut mu-
cosal immunization against lentivirus infection in NHP. Direct
mucosal introduction of replication-defective vectors offers an
alternative approach to address questions of immunogenicity
and efficacy in this model. Although cellular responses were
robust after intraileal injection, we did not detect a significant
level of IgG or IgA antibody from fecal extracts (data not
shown). Some animals showed IgA responses from the vaginal
7172 WANG ET AL. J. VIROL.
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9. washes, but the responses were low and inconsistent. Though
collection methods for mucosal secretions can be optimized
(19), it is likely that these responses are low in magnitude in
the GI tract.
While the results reported here describe the immunogenicity
of rAd5- and rAd35-based vectors, we have previously ana-
lyzed rAd41, an enteric tropic Ad, for its ability to generate
immune responses through direct ileum lumenal administra-
FIG. 5. Transduction efficiencies and immune responses induced by rAd35 and rAd35/41 chimeras. (A) Transduction of ileum explants by rAd
vectors. RLU, relative luminescence units. (B and C) Immune responses induced by rAd vectors in mice. Mice were administered the indicated
rAd vectors or empty rAd5 vector (Ϫ) via the intraileal route and boosted i.m. 3 weeks later. (B) Percentages of HIV-specific H-2Dd
/PA-9ϩ
CD8ϩ
T cells from spleens and small intestines 2 weeks after the i.m. rAd5-gp140B boost. (C) HIV-1-specific IFN-␥-secreting CD4ϩ
or CD8ϩ
T
lymphocytes from spleens 2 weeks after i.m. rAd5-gp140B boost. The data from each individual mouse and the mean values from the five mice
(black bars) are shown. -, empty vector. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001.
VOL. 83, 2009 INTRAILEAL rAd5 VACCINE STIMULATES MUCOSAL IMMUNITY 7173
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10. tion (17). When rAd41 was administered directly to the lumen
of the ileum, it efficiently primed antigen-specific cellular im-
munity. Ad41, a member of the Ad species F, has been re-
ported to bind/uptake more efficiently than Ad5 in the isolated
intestinal loops of rats. Furthermore, gene delivery with Ad5 to
differentiated enterocytes in vitro and to the rat jejunum in
vivo is extremely inefficient (7, 8, 44). However, the immune
responses induced by Ad41 and Ad5 through intraileal injec-
tion priming and i.m. boosting were not significantly different,
which highlighted that vectors derived from mucosal patho-
gens, both enteric and respiratory, can be used in these regi-
mens. However, due to the prevalence of neutralizing antibod-
ies to Ad5 and Ad41 in humans, the development and use of
alternative serotypes of Ad vectors for virus vaccines remain a
priority. Ad35 is one relatively well-characterized rare serotype
vector and has been associated with infections of the kidney
and urinary tract. Therefore, the fiber swap study was done for
two reasons: (i) to define the contribution of fiber versus hexon
to transduction of mucosal epithelium and (ii) to generate
vectors resistant to anti-Ad41 hexon neutralizing antibodies,
should they be mediated by fiber. Here, we found that rAd35
did not transduce the organ cultures of intestinal segments as
efficiently as rAd5 and that rAd35 was less immunogenic in the
ileal priming regimen, thus allowing us to address these ques-
tions. Unexpectedly, grafting the long fiber or the short fiber of
Ad41 to the rAd35 vector did not improve the transduction
efficiency of intestinal explants or immunogenicity, suggesting
that the Ad41 fiber is not the only determinant of enteric
tropism. However, these results suggest that the immunoge-
nicity of rAd vectors in vivo correlates with the efficiency of
transduction of intestinal explants ex vivo. We conclude that
the lower small intestine represents a target for antigen deliv-
ery with certain rAd vectors. Further development of special
formulations to deliver rAd to the ileum should be emphasized
for practical vaccination.
Our results suggest that it is important to pursue a mucosal
route of administration of rAd vaccine vectors for HIV. The
strong cellular immune responses induced by intraileal injec-
tion, further strengthened by i.m. boost, suggest a strategy for
protection of gut-associated lymphoid tissue lymphocytes from
HIV-1 infection. Possible mechanisms of immune stimulation
include persistent antigen expression from lamina propria cells
transduced by the rAd vector (7, 40). Further characterization
of the immune responses induced by the vaccination regimens
employed in this work, such as the balance of CD8ϩ
and CD4ϩ
T-cell responses, the polyfunctionality of the T cells, and the
duration of the immune response, could provide a better pre-
diction as to which is the most important type of immune
response for protection against HIV-1 (22). Regarding the
applicability of these data to other species, as well as the
uncertain applicability to humans, this study represents one of
a stepwise series of studies to determine whether mucosal
immunity can be enhanced by different rAd vectors with alter-
native routes of administration. Having been demonstrated
with rodents, this will next be evaluated with nonhuman pri-
mate challenge models to determine its contribution to im-
mune protection. Should these results support the concept of a
mucosal vaccine, an effort will be made to formulate an Ad
vector for delivery to the ileum in humans, since the site-
specific delivery technology to deliver drugs and vaccines to the
ileum has been developed with partial success so far (14, 16,
27, 36).
The use of rAd5 vaccines in populations with moderate to
high neutralizing antibodies to Ad5 based on the STEP trial
(3a) results is a concern. It will be difficult to define mecha-
nisms responsible for this effect or even to document it defin-
itively. At the same time, rAd5 vectors are well characterized
and induce strong immune responses. There are numerous
differences in vector design, insert combination, and immuni-
zation regimen that may result in different performances in
future clinical studies. Regardless of its future clinical utility,
rAd5 remains a useful model vector for inducing mucosal im-
munity and for determining how mucosal immune responses
contribute to protection. Once these key questions are ad-
dressed, rAd5 vectors can be used if justified by efficacy/safety
considerations, or, if concerns remain, other vectors can be
substituted.
ACKNOWLEDGMENTS
We thank Ati Tislerics for help with manuscript preparation, Brenda
Hartman and Morteza Loghmani for assistance with figures, Srinivas
Rao and Saran Bao for help with surgical techniques, D. Margulies and
the NIAID Tetramer Core Facility for production of tetramers, and
members of the Nabel laboratory for helpful discussions.
This work was supported by the Intramural Research Program of the
National Institutes of Health, Vaccine Research Center, NIAID, and
by the Bill and Melinda Gates Foundation.
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