JOURNAL OF VIROLOGY, Jan. 2009, p. 748–756
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 2
Enhanced Induction of Intestinal Cellular Immunity by Oral Priming
with Enteric Adenovirus 41 Vectors?
Sung-Youl Ko,1Cheng Cheng,1Wing-Pui Kong,1Lingshu Wang,1Masaru Kanekiyo,1David Einfeld,2
C. Richter King,2Jason G. D. Gall,2and Gary J. Nabel1*
Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-3005, 40 Convent Drive, Bethesda,
Maryland 20892-3005,1and GenVec, Inc., 65 West Watkins Mill Rd., Gaithersburg, Maryland 208782
Received 28 August 2008/Accepted 27 October 2008
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal
T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal
mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have
evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and
systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41
expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based
vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env
followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8?T-cell response in the small
intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum
together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal
as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with
rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector
delivery for primary immunization facilitates the generation of mucosal immunity.
Most infectious pathogens enter the body through mucosal
surfaces. The mucosa therefore represents a first line of de-
fense against infection. HIV in particular invades through the
gastrointestinal, lower genital, and rectal mucosa (13). Several
studies indicate that human immunodeficiency virus (HIV)
depletes most of the CD4?T cells in gut-associated lymphoid
tissue in patients with AIDS. The virus targets CD4?effector
memory T cells bearing CCR5 (CD4?CCR5?TEM) in extra-
lymphoid effector sites in the intestinal mucosa, as well as
below and within the gastrointestinal tract epithelial layers. It
infects TEMcells through a receptor and coreceptor and in-
duces the apoptosis of target cells. This depletion of TEM
occurs predominantly within the first few weeks after HIV
infection (7, 20–22, 25, 26), so the early establishment of mu-
cosal immunity, especially in the gut, is important for protec-
tion against HIV infection.
Adenovirus 41 (Ad41) is a human serotype F Ad that exhib-
its tropism for the gastrointestinal tract. It is associated with
gastrointestinal disease: an estimated 2 to 6% of gastroenteritis
cases are caused by this virus (2, 6, 10, 37). Ad41 possesses two
distinct fibers, long and short, which are present in the virion in
equal ratios (15). The long fiber, similarly to fibers of respira-
tory adenovirus Ad5, binds to the coxsackie and Ad receptor,
but the function of the short fiber is unknown (31, 34). Unlike
Ad5, Ad41 does not contain the integrin-binding RGD motif in
its penton base protein for entry to host cells (1).
Recently, replication-deficient recombinant Ad41 (rAd41) vec-
tor expressing HIV envelope protein (Env) has been developed
that stimulated HIV Env-specific humoral and cellular immune
responses after prime and boost immunizations. Heterologous
prime-boost with rAd41-rAd5 immunization induced significantly
higher levels of cellular immune responses systemically than
rAd5-only vector-based immunization through an intramuscular
route of administration (18). In this study, we characterized a
rAd41 vector expressing HIV Env for its ability to transduce
different cell types in vitro and evaluate this vector in different
vaccination regimens, specifically analyzing whether priming by
the systemic response.
MATERIALS AND METHODS
Animals. Six- to 8-week-old female BALB/c mice were purchased from NCI/
DCT (Frederick, MD) and housed in the experimental animal facility of the
Vaccine Research Center, National Institute of Allergy and Infectious Diseases
(NIAID), NIH (Bethesda, MD). All animal experiments were reviewed and
approved by the Animal Care and Use Committee, Vaccine Research Center,
NIAID, NIH, and were performed in accordance with all relevant federal and
NIH guidelines and regulations.
DNA and rAd vaccines. The VRC2805 plasmids expressing gp145?CFI?V1V2
of HIV-1 clade B and rAd5 or rAd41 expressing gp140?CFI?V1V2 of HIV-1
clade B (rAd5-Env or rAd41-Env) were prepared as described previously (9, 18).
Cell line and dendritic cell (DC) transduction with rAd vectors. All cell lines
were obtained from the American Type Culture Collection (Manassas, VA) and
grown in the recommended media. The cells were plated in 96-well plates
overnight and then transduced with rAd vectors encoding luciferase at the
indicated titers for 1 h in medium containing 2% fetal bovine serum (FBS). The
transduced cells were grown in fresh medium containing 10% FBS for 24 h after
transduction and assayed using a luciferase assay kit (Promega Corporation,
Immunization. For Ad only, mice were immunized with 109virus particles
(VP) of rAd-Env by the intramuscular or oral route. For DNA prime/rAd boost
immunizations, mice were primed with an intramuscular injection of 50 ?g of
DNA into the hind leg three times at 2-week intervals and boosted intramuscu-
larly with 109VP of rAd-Env 2 weeks later. For rAd prime/rAd intramuscular
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-
3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: (301)
496-1852. Fax: (301) 4480-0274. E-mail: email@example.com.
?Published ahead of print on 5 November 2008.
at NIH Library on December 29, 2008
boost immunizations, 109VP of rAd-Env was delivered either orally (by oral
gavage) or intramuscularly, and animals received an intramuscular boost with 109
VP of rAd5-Env 3 weeks later. The immunized mice were sacrificed 3 weeks after
the Ad-only immunization or 2 weeks after the boost immunization. Mice were
fasted overnight before oral immunizations.
Ileal injections. Following 16 h of fasting, mice were anesthetized using ket-
amine-xylazine (25 and 5 mg per kg of body weight, respectively) administered
intramuscularly. The animals were carefully monitored and kept warm through-
out the surgical procedure. Using an aseptic technique, a midline abdominal
incision was made, and the ileum was readily identified by locating the cecum.
Two small atraumatic serrated prewetted clamps (catalog no. 18055-02; Fine
Science Tools) were placed at the ileum-cecum junction and 5 cm upstream of
the cecum. VP (1010) in a total volume of 0.1 ml of rAd41 vector encoding HIV-1
gp140B (rAd41gp140B) was injected into the isolated ileum. Following 20 min of
incubation, the clamps were released and the abdominal cavity was closed. The
animals then received 0.05 mg/kg buprenorphine for discomfort following the
surgery. Gene expression and immunogenicity were determined after the ileal
injection of rAd5 encoding luciferase or rAd5 encoding HIV-1 gp140B, respec-
tively, with and without clamping, and the optimal time for the vector to infect
intestinal cells was determined. Transgene expression measured by luciferase
activity was higher in the groups with clamps, and 20 min was sufficient to infect
intestinal cells (data not shown), indicating that clamping facilitated the contact
of virus with the intestinal cells by separating the vector from digestive contents
and by distending the ileum.
Isolation and culture of DC from BM and spleen. Bone marrow (BM)-derived
DC were obtained from the BM of BALB/c mice and cultured as previously
described (14). More than 80% of these cells incubated in the presence of murine
granulocyte-macrophage colony-stimulating factor for 1 week expressed DC
surface markers CD11b and CD11c, as measured by flow cytometry. Lymphoid
DC (CD8?DC) and plasmacytoid DC (B220?DC) were isolated from mouse
spleens by magnetic cell sorting according to the manufacturer’s instructions
(Miltenyi Biotec, Auburn, CA). More than 90% of these purified cells expressed
CD8 or B220, as measured by antibody staining of the cells.
Lymphocyte preparation. Peripheral blood mononuclear cells (PBMC) were
purified using Lympholyte (Cedarlane Laboratories Ltd., Burlington, Ontario,
Canada) according to the manufacturer’s instructions. Single cells from mouse
spleen and mesenteric lymph nodes (MLN) were separated by mincing them
using a nylon mesh screen. For the measurement of cellular immune responses
in the small intestine, total lymphocytes (intraepithelial and lamina propria
lymphocytes) from the jejunum were prepared at the same time by enzyme
digestion. Briefly, Peyer’s patches were removed and the small intestine was
opened longitudinally. The intestine was flushed with phosphate-buffered saline
(PBS) and cut into 2-mm-long pieces in an enzyme solution of RPMI 1640
containing 50 mg/100 ml collagenase II (Sigma, St. Louis, MO) and 10% FBS
and then incubated at 37°C in a shaking incubator at 300 rpm for 30 min.
Lymphocytes in the supernatant were purified by 40 and 75% Percoll gradient
centrifugation at 1,200 ? g for 15 min, and the purified lymphocytes were
collected from above the 75% Percoll layer.
Tetramer staining of antigen-specific CD8 cells. Lymphocytes or PBMC were
stained with phycoerythrin-conjugated Dd/PA9 tetramer and then fluorescein
isothiocyanate-conjugated anti-mouse CD3 monoclonal antibody (MAb) (clone
145-2C11; BD Pharmingen), peridinin chlorophyll protein-Cy5.5-conjugated
anti-mouse CD8? MAb (clone 53-6.7; BD Pharmingen), and allophycocyanin-
conjugated anti-mouse CD19 MAb (clone 6D5; Biolegend). The stained cells
were examined by using a BD LSR-II (BD Pharmingen), and the data were
analyzed by FlowJo software (Tree Star Inc.).
ELISA detection of antibodies to HIV Env. HIV gp140B-specific immunoglob-
ulin G (IgG) and IgA were examined as follows: 96-well enzyme-linked immu-
nosorbent assay (ELISA) plates were coated with 2 ?g/ml recombinant HIV
gp140 clade B protein (VRC2801), incubated at 4°C overnight, and blocked with
PBS containing 1% bovine serum albumin at 37°C for an hour. Sera from the
immunized mice were diluted by twofold serial dilutions, and the diluted sera
were added. The plates then were incubated at 37°C for 2 h. Horseradish
peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoRseseach Lab-
oratories, Inc., West Grove, PA) and IgA (Southern Biotech, Birmingham, AL)
were added and incubated at 37°C for an hour. 3,3?,5?,5-Tetramethylbenzidine
(TMB; Sigma) in HRP substrate was added to each well, and yellow color that
developed after the addition of 0.5 M H2SO4was measured at 450 nm.
Neutralization assays. Sera from immunized mice were inactivated by being
heated at 56°C for an hour and diluted with RPMI 1640 medium with 2% FBS
and then mixed with the indicated rAd vector encoding luciferase for an hour at
room temperature. The neutralized virus was used to infect 293 cells (at a
multiplicity of infection of 100) based on the number of VP per cell for 2 h, and
then the medium was replaced with RPMI 1640 containing 10% FBS and incu-
bated overnight at 37°C. The infected 293 cells were lysed, and the supernatants
were mixed with luciferin. The luminescence was measured using a microplate
scintillation and luminescence counter (PerkinElmer, Shelton, CT).
Data and statistical analysis. Results are expressed as means ? standard errors.
Statistical analyses were performed upon comparisons made between the control
groups and treated groups or between treated groups using Student’s t test.
Transduction of a variety of cell types in vitro by rAd41.
rAd41 vectors encoding luciferase were prepared, and their
ability to transduce different cell types was analyzed in vitro
(Fig. 1). The rAd41 vector transduced 293T cells with higher
efficiency than an rAd5 vector. It also readily transduced hu-
man intestinal cell lines, FHs74 intestine and Caco-2 cells (Fig.
1A). In addition to intestinal epithelial cells, its lymphoid tro-
pism was analyzed on murine lymphoid and plasmacytoid DC
isolated from a mouse spleen and myeloid DC derived from
mouse BM. Although the transduction efficiencies of the latter
cells were lower than that of the rAd5 vector, gene transfer was
readily observed (Fig. 1B). rAd41 therefore was able to deliver
transgenes into multiple cell types of epithelial and lymphoid
Comparable antigen-specific cellular immune responses by
rAd41 and rAd5 vectors. To assess whether rAd41 vectors can
induce immune responses comparable to those of rAd5 vectors
after a single intramuscular immunization, mice were immu-
nized intramuscularly with rAd41-Env or rAd5-Env. The im-
munized mice were sacrificed 3 weeks later, and Env-specific
CD8?T cells were examined in PBMC using a tetramer to an
FIG. 1. Transduction of different cell lines and mouse DC with
rAd5 and rAd41. (A) All cell lines were obtained from the ATCC and
were plated in 96-well plates. (B) Mouse DC were isolated as described
in Materials and Methods. Cells were transduced with rAd5 or rAd41
encoding luciferase at the indicated multiplicity of infection (MOI)
based on viral particles, and luciferase expression was measured 24 h
after transduction. BMDC, DC derived from mouse BM.
VOL. 83, 2009GUT-TROPIC rAd41 VACCINE STIMULATES MUCOSAL IMMUNITY749
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immunodominant epitope in the V3 loop, H-2Dd/PA9, a re-
sponse that corresponded to functional activation, since cells
showed an increase in intracellular cytokine staining for inter-
leukin-2, gamma interferon, and tumor necrosis factor alpha
after stimulation with the same peptide in vitro (M. Honda, R.
Wang, W. 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 pub-
lication). Representative flow plots confirmed tetramer-spe-
cific CD8 responses for each vector (Fig. 2A), and rAd41
vaccine-immunized mice showed levels of Dd/PA9?CD8?T
cells that were similar to those of rAd5 vaccine-immunized
mice (Fig. 2B). The antibody response also was measured by
the detection of serum IgG ELISA titers. Significantly higher
levels of HIV Env-specific IgG were detected in rAd41-Env-
immunized animals than in controls, though titers were lower
than those in rAd5 vaccine-immunized mice (Fig. 2C). An
examination of the IgG and IgA responses in vaginal washes
and fecal extracts revealed minimally detectable responses
(data not shown) despite the systemic effects, suggesting alter-
native mechanisms of stimulation of these disparate compart-
ments. These results demonstrate that the rAd41 vector in-
immune responses after a single intramuscular immunization.
rAd41 vectors boost immune responses primed by DNA vac-
cination. Although rAd5 vectors are known to readily boost
DNA-primed animals (32), the ability of rAd41 vectors to
boost DNA-primed responses is not known. To address this
question, mice were immunized intramuscularly with DNA
encoding HIV Env three times at 2-week intervals and boosted
intramuscularly or orally immunized with rAd41-Env or rAd5-
Env 2 weeks later. Two weeks after boosting, immunized mice
were sacrificed; HIV-1 Env-specific CD8?T cells were exam-
ined in PBMC, spleen, MLN, and the small intestine; and HIV
Env-specific IgG titers were measured in serum. Representa-
tive flow plots confirmed tetramer-specific responses in both
spleen and intraepithelial intestinal lymphocytes (Fig. 3A). In
both systemic and intestinal tissues, mice boosted orally with
rAd5-Env or rAd41-Env did not generate substantial Env-
specific Dd/PA9?CD8?T-cell tetramer responses (Fig. 3A,
columns 5 and 6). However, mice that were boosted with rAd
vectors intramuscularly showed substantially higher levels of
cellular immune responses than the negative control group in
both compartments (Fig. 3B, compare lanes 3 and 4 to lane 1),
and both rAd41 and rAd5 vaccine-boosted groups elicited sig-
nificantly higher tetramer responses than single rAd5 vaccine-
immunized mice in the spleen (Fig. 3B, spleen panel, compare
lanes 3 and 4 to lane 2). These results indicate that the rAd41
vaccine induced cellular immunity similar to that induced by
rAd5 in both systemic and mucosal compartments after DNA
prime/intramuscular rAd vaccine boost immunization. The
IgG analysis revealed differences in the antibody response be-
tween the two vectors. rAd41 vectors stimulated a substantial
increase in ELISA titers, and they were greater after intramus-
cular than after oral boosting (Fig. 3C, compare lane 4 to lane
6), but rAd5 stimulated these responses more effectively (Fig.
FIG. 2. rAd41 vector induced cellular immune responses comparable to those of rAd5 vector with a single intramuscular immunization. Mice
were immunized once with rAd5-Env or rAd41-Env by the intramuscular route and sacrificed 3 weeks later. (A) Representative flow plots of
Dd/PA9 tetramer staining for each vector are shown. SSC, side-scatter characteristics. (B) The numbers of HIV Env-specific Dd/PA9?CD8 cells
were examined in PBMC by flow cytometry. (C) HIV gp140-specific IgG titers were measured in serum. Bars represent the mean values.*, P ?
0.02;**, P ? 0.01.
750KO ET AL. J. VIROL.
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FIG. 3. rAd41 can substitute for rAd5 as a vector for DNA priming-rAd boosting. Mice were injected intramuscularly with DNA three times
at 2-week intervals and intramuscularly or orally boosted 2 weeks later with rAd5-Env or rAd41-Env. Two weeks later, the mice were sacrificed.
(A) Representative flow plots of Dd/PA9 tetramer staining for the indicated immunizations are shown. SSC, side-scatter characteristics; i.m.,
intramuscular; p.o., per os. (B) The numbers of HIV Env-specific Dd/PA9?CD8?T cells were examined in PBMC, spleen, MLN, and the small
intestine by flow cytometry. C, control. (C) The levels of HIV Env-specific IgG were examined in serum. Bars represent the mean values.*, P ?
0.05;**, P ? 0.02;***, P ? 0.01.
VOL. 83, 2009GUT-TROPIC rAd41 VACCINE STIMULATES MUCOSAL IMMUNITY751
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3C, compare lanes 3 and 5 to lanes 4 and 6). Interestingly,
rAd41 and rAd5 vaccine-boosted mice using the oral route of
administration produced significantly higher levels of IgG than
the negative control mice (Fig. 3C, compare lanes 5 and 6 to
lane 1) despite the fact that the vaccinations did not induce
high levels of cellular immune responses (Fig. 3B, lanes 5 and
6). Taken together, these data indicate that following DNA
priming, rAd41 and rAd5 vectors induce comparable cellular
immune responses in both mucosal and systemic compart-
ments and stimulate systemic antibody responses.
Oral immunization with rAd41 vaccine in PBS is more ef-
fective than that in sodium bicarbonate. The low pH of the
stomach represents a barrier for the introduction of rAd and
other vaccine vectors for oral immunization. To determine
whether different diluents could affect the efficacy of vaccina-
tion, we compared a high-pH solution with low buffering ca-
pacity, sodium bicarbonate, to a neutral-pH diluent with higher
buffering capacity for their ability to stimulate rAd-mediated
cellular and humoral immune responses. A single oral immu-
nization did not elicit HIV Env-specific cellular immune re-
sponses in the tetramer assay (Fig. 4A), so we boosted orally
immunized mice with rAd5-Env intramuscularly. Mice primed
orally with rAd41-Env in PBS produced higher cellular im-
mune responses in the small intestine than those of bicarbon-
ate, although no differences were noted in the spleen (Fig. 4B,
lanes 2 and 3). These findings suggest that PBS is the preferred
diluent to prime for an rAd41-stimulated mucosal immune
Heterologous rAd41 prime/rAd5 boost of cellular immunity
in the small intestine as well as humoral immunity. We next
compared rAd41 to rAd5 for its ability to prime an rAd5
vaccine boost. Intramuscular priming with rAd41-Env induced
an increased antigen-specific CD8?T-cell response in both
systemic and mucosal compartments compared to that of
rAd5-Env (Fig. 5A, lanes 5 and 6). Notably, oral priming with
rAd41-Env also stimulated a higher-magnitude response than
rAd5-Env, more so in the small intestine than the spleen (Fig.
5A, lanes 3 and 4). Similarly, intramuscular rAd41/rAd5 elic-
ited the highest-titer ELISA responses, which were greater
than those of intramuscular rAd5/rAd5 or by oral priming with
FIG. 4. PBS diluent is more effective than sodium bicarbonate for the induction of mucosal cellular immune responses in the small intestine
for oral delivery. (A) Mice were orally primed with rAd5-Env or rAd41-Env alone or (B) followed by an intramuscular (i.m.) boost with rAd5-Env.
p.o., per os; C, control. The numbers of HIV Env-specific Dd/PA9?CD8 cells were examined in the spleen and the small intestine by flow
cytometry. Bars represent the mean values.*, P ? 0.01.
752KO ET AL. J. VIROL.
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either vector (Fig. 5B). These results demonstrate that priming
with rAd41 vectors induced CD8?T-cell immunity in the small
intestine by an oral route as well as stimulated systemic IgG
responses to a greater extent than did intramuscular priming.
Generation of vector-neutralizing antibodies by alternative
routes of immunization. Since priming with rAd vectors may
generate vector-specific antibodies that can affect the efficiency
of the subsequent rAd boosts, we analyzed mouse sera for the
presence of neutralizing antibodies after the administration of
Ad vectors (Fig. 5C). The intramuscular administration of
rAd5 vectors elicited antibodies that specifically neutralized
rAd5 vector transduction by more than 100-fold compared to
that of nonimmune mouse sera (Fig. 5C, left, compare bars 1
and 5). As expected, this sera did not neutralize a rAd41
reporter vector (Fig. 5C, right, bar 1), consistently with the
known designation of these viruses as distinct serotypes. Sim-
ilarly, the intramuscular administration of rAd41 vectors elic-
ited rAd41-specific neutralizing antibodies that did not neu-
tralize rAd5 vectors (Fig. 5C, left, bar 2). Notably, the oral
administration of Ad5 did not elicit neutralizing antibodies to
FIG. 5. Stimulation of cellular immunity in the small intestine by rAd41 oral prime-rAd5 intramuscular boost vaccination and decreased vector-
specific neutralizing antibodies by oral compared to intramuscular immunization. Mice were primed orally or intramuscularly (i.m.) with rAd5-Env or
rAd41-Env diluted in PBS and boosted intramuscularly with rAd5-Env. p.o., per os; C, control. (A) The numbers of HIV Env-specific Dd/PA9?CD8?
T cells were examined in the spleen (left) and the small intestine (right) by flow cytometry. (B) HIV Env-specific IgG titers were assessed in serum.
(C) Mice were immunized with rAd5-Env or rAd41-Env intramuscularly or orally. Three weeks after immunization, the sera from vaccinated or naive
were added to 293 cells at a multiplicity of infection of 100. After incubation, luminescence from Ad5-luciferase-infected (left) and Ad41-luciferase-
infected (right) 293 cells were measured. Bars represent the mean values.*, P ? 0.05;**, P ? 0.02;***, P ? 0.01.
VOL. 83, 2009GUT-TROPIC rAd41 VACCINE STIMULATES MUCOSAL IMMUNITY753
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the homologous vector (Fig. 5C, left, bar 3). Similarly, the oral
administration of Ad41 did not stimulate the production of
neutralizing antibodies to Ad41 (Fig. 5C, right, bar 4). These
results suggest that heterologous prime/boost vaccination reg-
imens circumvent vector-induced immunity from priming and
that oral immunization, while less potent than intramuscular
priming, can prime for a secondary immunization without in-
ducing systemic antivector humoral immunity.
Direct injection of rAd41 into ileum. To circumvent the
degradation of vectors by low gastric pH or the presence of
degradative enzymes in the stomach or small intestine, we
directly injected rAd41-Env into the ileum surgically. Three
weeks later, animals received a booster vaccination with an
intramuscular rAd5 vector. The injection of rAd41-Env alone
into the ileum induced a substantial Dd/PA9?CD8 T-cell
response in PBMC prior to boosting (Fig. 6A). After the boost
with intramuscular rAd5-Env, it induced remarkably high lev-
els of CD8?T cells in the spleen (Fig. 6B) and especially high
levels in the small intestine, presumably a mix of both intra-
epithelial and laminar propria lymphocytes (Fig. 6C). These
results indicate that the direct delivery of rAd41 into the lower
small intestine effectively primes mucosal and systemic cellular
Due to the rapid development of antibody neutralization
escape mutations (29) or through its ability to evade neutral-
ization because of glycan shielding (38), HIV has evaded at-
tempts to control infection by eliciting a humoral immune
response. The potential importance of CD8?T cells in the
control of HIV infection has been demonstrated in a variety of
studies. Simian immunodeficiency virus (SIV) replication is
markedly increased in monkeys when CD8?lymphocytes are
depleted (33), and SIV-specific central memory CD8?T cells
and linked CD4?T cells are responsible for protection against
disease progression in a monkey model (36). It is well known
that replication-deficient virus-based vaccines predominantly
induce cytotoxic T lymphocytes and CD4?T-cell responses
(24) and that rAd5-based vectors are among the most potent
stimulators of systemic responses (35). Such vectors have been
tested in intramuscular immunization alone and in DNA-rAd
vaccine regimens in human clinical trials (8, 12, 27). Due to the
high prevalence of neutralizing antibodies against Ad5, alter-
native serotypes and other vectors are being actively pursued.
Here, we evaluated a rAd41 vector to determine if it can
prime for a rAd5 boost immunization and evaluated its ability
to induce cellular immune responses in the gut by different
routes of delivery. We have previously demonstrated that
rAd41 prime/rAd5 boost was more effective than rAd5 prime/
rAd41 boost in stimulating cellular immunity (18). In the
present study, we examined the route of the administration of
Ad41 vector as a priming agent. rAd41-Env induced similar
cellular immune responses to rAd5-Env after both a single
intramuscular immunization and DNA prime/Ad boost immu-
nization, which suggests that rAd41 is a good substitute for
rAd5 in systemic immunization for the induction of cellular
immune responses. In in vitro studies, sera from intramuscular
rAd5-immunized mice, but not orally immunized mice, dis-
played neutralizing activity against rAd5. Similar results were
observed with a rAd41-based regimen with respect to Ad41
neutralization (Fig. 5C). The effect of the neutralizing activity
could be seen in the lack of a T-cell boost in the Ad5 homol-
ogous intramuscular prime-boost group (Fig. 5A). However,
this same regimen induced significantly higher humoral im-
mune responses than single intramuscular rAd5 vaccine immu-
nization (Fig. 5B). Intramuscular administration with low-dose
vector stimulated humoral immune responses to the transgene
that could be boosted further despite the presence of anti-
vector neutralizing antibodies in the homologous prime-boost
immunization. These results are in agreement with those
generated in a Dengue virus vaccine model (28).
Several approaches have been taken to elicit immune re-
sponses against HIV infection in mucosal compartments, in-
cluding intestinal, vaginal, and rectal mucosa. Intranasal im-
munization with a rAd5 vaccine generated stronger IgA
FIG. 6. Ileal injection with rAd41-Env increases CD8 cell responses in both systemic and mucosal compartments. Mice were directly primed
with rAd41-Env by ileal surgery as described in Materials and Methods. (A) The numbers of HIV Env-specific Dd/PA9?CD8?T cells were
examined in PBMC by flow cytometry 3 weeks after the immunization. The primed mice were boosted with rAd5-Env intramuscularly (i.m.). The
numbers of HIV Env-specific Dd/PA9?CD8 cells were examined in the spleen (B) and the small intestine (C) by flow cytometry 2 weeks later.
p.o., per os. Bars represent the mean values.*, P ? 0.05;**, P ? 0.02;***, P ? 0.01.
754KO ET AL. J. VIROL.
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responses in systemic and mucosal compartments than intra-
muscular immunization in mice, but safety concerns may limit
the use of this route of administration for rAd5 vaccines (19).
Intrarectal immunization with peptide vaccines composed of
HIV or SIV antigens with adjuvant can induce mucosal cyto-
toxic T lymphocytes more efficiently and control RNA levels
better in mucosal and systemic compartments than subcutane-
ous immunization in macaques (4). Oral priming with enteric
coated rAd5-based HIV vaccines followed by intranasal boost-
ing with an envelope peptide cocktail with adjuvant induced
HIV-specific cellular immune responses in the intestine of
rhesus macaques (23). In addition, several studies have dem-
onstrated that the mucosal administration of replication-com-
petent Ad-based vaccines can generate immune responses
against HIV antigens. Intranasal priming with replication-com-
petent Ad4, Ad5, and/or Ad7 vaccines followed by intramus-
cular boosting with Env elicited mucosal immune responses
and protection against HIV challenge in chimpanzees (30).
Intranasal-oral or oral-oral priming with a replication-compe-
tent Ad5-HIV vaccine followed by a protein vaccine induced
mucosal immunity in rhesus macaques (40). These studies
demonstrate the possibility of generating mucosal and systemic
immune responses to HIV antigens, but the potency and vac-
cination regimens need to be further optimized before advanc-
ing these vaccines into human clinical trials.
Novel vectors that possess natural mucosal tropism may
have advantages over these reported vectors in terms of ad-
ministration, safety, and vaccine potency. When antigen or
vector is administered by an oral route, it typically is inefficient
compared to the efficiency of injection, often requiring 100- to
1,000-fold higher levels of protein or virus due to loss by
degradation or mucosal clearance. The actual delivered dose
therefore was likely much less than the injected amount. This
finding suggests that antigen delivery is relatively efficient, pos-
sibly due in part to the gut tropism of Ad41. In the present
study, oral rAd41-Env priming induced the highest HIV Env-
specific CD8?T-cell responses, as determined by a tetramer
response in the small intestine. Sera from orally immunized
animals did not block the entry of the homologous serotype
vectors in neutralization assays, indicating that oral rAd im-
munization induces either weak or no neutralizing antibodies
against the administered rAd vector. These findings are con-
sistent with studies of a different route of delivery, intranasal
inoculation, which also does not elicit Ad5 neutralizing anti-
body (39). It is widely accepted that the systemic immunization
of protein- or peptide-based vaccine induces only systemic
immune responses, whereas mucosal immunization induces
mucosal as well as systemic immune responses (3, 16). In this
study, heterologous oral rAd41 prime-intramuscular rAd5
boost induced the highest levels of antigen-specific tetramer-
positive CD8?T-cell responses in the small intestine, whereas
heterologous intramuscular rAd41-intramuscular rAd5 boost
induced the highest systemic response. The increased cellular
immune responses by heterologous intramuscular rAd41 prim-
ing-intramuscular rAd5 boosting may be related not only to the
evasion of preexisting immunity to rAd41 but also to intrinsic
characteristics of the virus vector. Possibly, antigen-presenting
cells activated by intramuscular rAd41 vaccination educate im-
mune cells, including T cells, in the draining lymph nodes and
direct these cells to the mucosal compartment in the gut. The
microenvironments of various secondary lymphatic tissues are
very different: antigen-presenting cells of the same phenotype
are able to respond differently to the antigen based upon where
the immune responses are initiated. For example, CD8??DC
present orally delivered antigens in MLN, while CD8??DC in
the spleen present intravenously delivered antigens (11). The
types of antigens, in addition to immune cells, also affect the
nature of the reaction. CD8??DC mediate antiviral immunity
to viral infections by subcutaneous or intravenous infections,
whereas CD8??DC are activated against protein antigen with
adjuvant delivered intranasally (5, 17). rAd41 vector vaccines
delivered to the intestinal mucosa likely utilize such mecha-
nisms to induce mucosal cellular immune responses in the gut
in combination with Ad5 boosting and represent a potential
approach to elicit protective immune responses to HIV. While
direct injection into the ileum represents an experimental tool
to demonstrate this effect, the development of appropriate
formulations such as enteric coatings or nanoparticles could
allow the vector to avoid the degradative environment of the
upper gastrointestinal tract and be amenable to clinical appli-
We thank Ati Tislerics for assistance with manuscript preparation,
Brenda Hartman for figure preparation, members of the Nabel labo-
ratory for helpful discussions and advice, and Srinivas Rao and col-
leagues for help with anesthetic and surgical techniques.
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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