JOURNAL OF VIROLOGY, Jan. 2010, p. 630–638
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 1
Differential Specificity and Immunogenicity of Adenovirus Type 5
Neutralizing Antibodies Elicited by Natural Infection
Cheng Cheng,1Jason G. D. Gall,2Martha Nason,1C. Richter King,2† Richard A. Koup,1
Mario Roederer,1M. Juliana McElrath,3Cecilia A. Morgan,3Gavin Churchyard,4
Lindsey R. Baden,5Ann C. Duerr,3Michael C. Keefer,6
Barney S. Graham,1and Gary J. Nabel1*
Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-3005, 40 Convent Drive, Bethesda,
Maryland 20892-30051; GenVec, Inc., 65 West Watkins Mill Rd., Gaithersburg, Maryland 208782; HIV Vaccine Trials Network,
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. NE, LE-5034, Seattle, Washington 981093;
Aurum Health Research, KOSH, P.O. Box 61587, Marshalltown 2107, South Africa4;
HIV Vaccine Trials Network, Brigham and Women’s Hospital, Infectious Disease,
PBB-A4, 75 Francis St., Boston, Massachusetts 021155; and University of
Rochester School of Medicine & Dentistry, 601 Elmwood Ave.,
Box no. 689, Rochester, New York 146426
Received 29 April 2009/Accepted 5 October 2009
A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors
showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus
type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and
vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether
subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects
receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their
Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing
antibodies were directed to different components of the virion, depending on whether they were elicited by natural
infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were
directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid
proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced
the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing
antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5
vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future
AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.
Recombinant adenovirus (rAd)-based vectors are currently
under investigation in a variety of gene therapy and T-cell-
based vaccine clinical trials. There are more than 370 such
ongoing clinical trials for broad applications, including infec-
tious diseases and cancer therapy (http://www.wiley.co.uk
/genetherapy/clinical/). Based on supportive data from nonhu-
man primate studies, rAd-based vectors have been developed
and tested in human clinical trials to deliver human immuno-
deficiency virus (HIV-1) gene products that stimulate HIV-
specific immune responses. Preexisting immunity to Ad sero-
type 5 (Ad5), from which most vectors are derived, is common
in humans. Though neutralizing antibodies to Ad5 may reduce
the immunogenicity of Ad5-based vectors in animal models
(16), their effect on immunity in subjects with previous Ad5
infection is poorly understood. In the STEP trial, which tested
a Merck rAd5 vaccine encoding HIV-1 Gag, Pol, and Nef,
vaccination failed to show protection, either by lowering viral
load or by decreasing acquisition of infection (3, 9, 12, 21).
Furthermore, the possibility was raised that subjects with pre-
existing neutralizing antibodies from natural Ad5 infection
may have carried an increased risk of HIV infection after
vaccination. Thus, understanding the nature and immune ef-
fects of Ad5 seropositivity in humans is important to the de-
velopment of vaccines against AIDS and other diseases.
Ad5 is a common cause of respiratory disease and an occa-
sional cause of gastroenteritis in humans, and exposure before
adolescence is common in human populations (19). Such ex-
posure stimulates both innate and adaptive immune responses
that generate neutralizing antibodies and virus-specific T-cell
responses (6). These antibodies can also synergize with each
other to achieve maximum viral neutralization (7, 22). The
capsid protein specificity of Ad5 neutralizing antibodies has
been reported for humans following administration of rAd5
gene therapy vectors for advanced liver or lung cancer (7, 10).
However, results were presented solely for antibodies induced
by administration of rAd5. One report has assessed Ad5 neu-
* 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) 480-0274. E-mail: email@example.com.
† Present address: International AIDS Vaccine Initiative, AIDS
Vaccine Design and Development Laboratory, Brooklyn Army Termi-
nal, 140 58th St., Brooklyn, NY 11220.
?Published ahead of print on 21 October 2009.
by Jason Gall on January 15, 2010
tralizing antibodies with a healthy human population that was
Ad5 seropositive from natural exposure to the virus (18). The
median titer of the population was presented, but the fre-
quency of protein-specific neutralizing antibody has not been
defined for humans.
Here we describe the first report of the natural frequency
and effect on immunization of neutralizing antibodies specific
for different Ad capsid proteins in human subjects. We address
the fundamental mechanisms of how humans generate neu-
tralizing antibodies to a common cold virus that is in wide-
spread use as a vector for gene therapy and vaccines. Such
mechanisms may also be applicable to other nonenveloped
viruses, including adeno-associated viruses and other viruses
containing multiple envelope surface proteins, like influenza.
To analyze the contribution of anti-capsid antibodies to neu-
tralization by different human serum samples, wild-type and
chimeric vectors were utilized. For example, a rAd type 5
(rAd5) vector with a fiber derived from Ad35 fiber (rAd5 F35)
can be used to analyze the anti-Ad5 capsid response indepen-
dent of fiber. Conversely, a rAd35 vector with a fiber trans-
posed from Ad5 can determine the specificity of neutralization
mediated by the Ad5 fiber. Using these vectors, we have ana-
lyzed human serum samples from two HIV vaccine clinical
trials, VRC 006 and HVTN 204, in which a single-dose rAd5
vaccine alone and a three-dose DNA prime/single dose rAd5
boost vaccine encoding HIV-1 Env A,B, and C; Gag; and Pol,
respectively, were administered. Thus, we sought to character-
ize the specificity of rAd5 neutralizing antibodies in Ad5-im-
mune subjects and to determine their effect on immune re-
sponses elicited by vaccination.
MATERIALS AND METHODS
Ad vector construction, production, and purification. Replication-deficient
rAd5 F35 and rAd35 F5 were generated in 293-ORF6 cells essentially as de-
scribed previously (2, 8, 11). The region encoding the Ad35 shaft and knob
(amino acids 45 to 323) was cloned into an Ad5 shuttle plasmid, pASE3(10)F35.
The region encoding the Ad5 shaft and knob (amino acids 46 to 581) was cloned
into the Ad35 shuttle plasmid pUC19.Ad35.F(5S.5K)_E4. The region of the fiber
protein that interacts with the viral capsid, the tail, was therefore homologous to
the capsid; this feature ensured that there was minimal perturbation of the virion
structure. The plasmids with the fiber-modified genes were recombined in Rec?
Escherichia coli with Ad5 or Ad35 vector-genome plasmids encoding an expres-
sion cassette for luciferase. The fiber-modified genome plasmid clones were
isolated through repeated bacterial transformation–colony formation. Fiber-
modified viral vectors were generated by transfection of 293-ORF6 cells, ampli-
fied, and purified as described previously (11). Replication-deficient rAd5 and
rAd35 were generated and produced using similar methods. The quality of the
rAd stocks was verified by growth kinetics, comparative analysis of transgene
expression, particle/infectious unit ratios, PCR analysis of the genomes, and
DNA sequencing of the modified regions.
Clinical trials. VRC006 is a Vaccine Research Center-sponsored phase I trial
described previously (4). A total of 36 healthy volunteers were divided into three
groups that were injected with 109, 1010, or 1011viral particles of Ad5-based
multivalent HIV vaccine vectors encoding HIV Envs from clades A, B, and C and
Gag and Pol from HIV clade B. Each group also included two persons injected
HVTN204 is a phase II trial to test a multivalent HIV DNA vaccine prime and
multivalent HIV Ad5 vector boost in human volunteers. The vaccine vectors
encode six HIV antigens, including three HIV Envs (Env clade A, Env clade B,
and Env clade C) and Gag, Nef, and Pol from HIV clade B. The volunteers were
given either four injections of placebo or three dosages of DNA vaccine at month
0, 1, and 2 and then given an Ad5 vector boost at month 6 (week 24).
Neutralization of adenovectors with human serum samples. The method for
analyzing the neutralization of adenovectors with human serum samples was
developed based on procedures published previously (17). A total of 127 samples
of prevaccinated human serum samples (week 0) from volunteers enrolled in the
TRIAD (HVTN204) HIV vaccine trial and 32 available prevaccination (week 0)
and postvaccination (week 4) serum samples from the VRC006 HIV vaccine trial
were obtained from the VRC immunology core laboratory. The serum samples
were inactivated by heating at 56°C for 60 min, and the inactivated serum
samples were diluted with 10% fetal bovine serum containing RPMI medium and
mixed with the indicated rAd vector encoding luciferase for 30 min at room
temperature for neutralization. To prepare A549 cell suspensions for transduc-
tion, A549 cells grown in a 75-cm2flask were harvested by treatment with 4 mM
EDTA and suspended in 10% fetal bovine serum containing RPMI medium. The
neutralized virus was used to transfect A549 cells at 100 PU/cell, and luciferase
expression was analyzed using the luciferase assay kit (Promega, Inc.) at 24 h
posttransduction. Serum samples that at the lowest dilution (1:36 due to limited
serum availability as noted) reduced viral transduction activity by more than 90%
were defined as seropositive (17). The neutralization titer for such seropositive
serum samples was also determined and defined as the maximum dilution that
can reduce the viral transduction by 90% as calculated using GraphPad Prism 5
Analysis of T-cell responses to vaccination. At 28 weeks after the first vacci-
nation (4 weeks post-rAd vaccination), the T-cell responses to individual HIV
antigen peptide pools were analyzed according to previously published methods
(4) by an intracellular cytokine staining (ICS) assay. The percentages of CD4?
and CD8?T cells producing cytokines were reported with background correc-
rAd5 HIV vaccine responders were identified by comparing ICS data with
antigen peptide stimulation and data without antigen peptide stimulation. A
composite definition was used to identify T-cell responses based on both statis-
tical criteria and the magnitude of the response. First, the proportion of positive
cells in response to the antigen must be statistically significantly different than the
proportion of positive cells in the background-stimulated sample at an ? of 0.01
by a one-sided Fisher exact test. In addition, the background-subtracted magni-
tude must be above a predefined antigen-specific threshold. The cutoff frequency
for a positive response was set at 0.045% for CD4?response to all HIV antigens,
0.070% for CD8?response to Env-C, 0.058% for CD8?response to Gag, and
0.045% for CD8?response to all other HIV antigens. Only if both of these
criteria are met is a sample considered to have a positive response to that
antigen. This definition is consistent with that used by the Vaccine Research
Center, NIH, in the analysis of other clinical trial data.
Statistical analysis. Serum samples were categorized as seropositive or seroneg-
ative for each of the four recombinant vectors, with a neutralization titer of more
than 36 categorized as positive. The subjects were also categorized into rAd5 HIV
vaccine responders or nonresponders as described above. Fisher’s exact (two-sided)
tests were used to calculate P values between subjects seronegative or seropositive
for antibodies to Ad5 and vaccine responder or nonresponder.
The magnitudes of CD4?or CD8?T-cell immune responses were compared
between seropositive and seronegative subjects using two-sided Wilcoxon rank sum
tests. These nonparametric tests were chosen due to the presence of large influential
points for some of the groups and to the moderate sample sizes for some of the
to false significance, a Bonferroni adjustment was used to define a threshold for
statistical significance of 0.001; P values above this threshold but below 0.05 are
noted in the text as marginally significant or suggestive of a trend.
Analysis of human sera for neutralization directed to dif-
ferent Ad5 proteins. Both Ad5 and Ad35 contain three major
surface proteins: fiber, hexon and penton base. These external
proteins play an important role in viral infection and are the
major targets of neutralizing antibodies in human sera. We
adapted a previously described assay (17) to differentiate be-
tween neutralization through fiber antibodies and through an-
tibodies against other capsid proteins. In the modified proto-
col, EDTA rather than trypsin was used to detach A549 cells
grown as a monolayer in a flask in order to avoid degradation
of cell surface receptors for Ad. Since the primary mechanism
by which Ad5 infects human cells is through direct interaction
between fiber and its primary receptor, coxsackie B, and Ad
receptor (CAR), we focused on the analysis of fiber-related
VOL. 84, 2010Ad5 IMMUNITY AND HIV-1 VACCINES631
by Jason Gall on January 15, 2010
and non-fiber-related neutralization. The panel of rAd vectors
included two chimeric vectors and the parental, unmodified
vectors (Fig. 1A) that showed similar transduction frequencies
on the A549 target cells (Fig. 1B). The rare serotype Ad35 was
used as the source for non-Ad5 capsid proteins, since serum
samples from North American populations rarely neutralize
Ad35. The Ad5 fiber was replaced with Ad35 fiber to generate
rAd5 F35, and this vector was used to detect neutralization of
Ad5 independent of fiber. In contrast, the complementary chi-
mera, rAd35 F5, rAd35 typed with Ad5 fiber, allowed the
FIG. 1. Recombinant and chimeric Ads determine the specificity of neutralizing antibodies in selected subjects. (A) Wild-type and chimeric
vectors based on Ad5 and Ad35 were used to analyze the neutralizing antibody activities against different viral capsid proteins. Fiber and other
major capsid proteins (including hexon and penton) in wild-type Ad5 contain neutralizing epitopes targeted by human antibodies. rAd5 F35 is an
Ad5-based chimeric vector that contains the fiber of Ad35 in place of Ad5 fiber. It contains all the Ad5 capsid proteins except fiber and was used
to analyze specific neutralizing antibodies to Ad5 capsid proteins excluding fiber. rAd35 F5 contains only one Ad5-specific neutralizing target, the
fiber. It was used to analyze specific neutralizing antibodies to Ad5 fiber. (B) Comparable transduction of A549 cells by different chimeric rAd
vectors. A549 cells were transduced with rAd vectors encoding luciferase (Ad5, Ad35, Ad5 F35, and Ad35 F5) or green fluorescent protein
(Control) at indicated multiples of infection viral particles (MOI). Luminescence was measured at 24 h after transduction. (C) Two examples of
human serum samples from the VRC006 trial that can neutralize Ad vectors. Human serum samples at week 0 (prior to vaccination) were diluted
as indicated, and their ability to neutralize adenoviral vectors was tested. Neutralization assays were performed in triplicate, and the average with
standard error is shown. Serum VRC006-12 could neutralize more than 90% of the infectivity of rAd5 and rAd35 F5 but not of rAd35 and rAd5
F35, showing primarily Ad5 fiber-specific neutralization. Serum VRC006-18 could neutralize more than 90% of the infectivity of rAd5, rAd5 F35,
and rAd35 F5 but not of rAd35, showing that Ad5 fiber and other capsid proteins were all targeted for neutralization.
632 CHENG ET AL.J. VIROL.
by Jason Gall on January 15, 2010
detection of Ad5 fiber-directed neutralization. The parental
viruses, rAd5 and rAd35, facilitated comparison of the poten-
cies of Ad5 immunity among subjects and excluded the possi-
bility of cross-reactive antibodies that might confound inter-
pretation of neutralization of the chimeras. Different patterns
of neutralization were observed. Serum from a representative
VRC 006 trial subject neutralized rAd35 F5 at high titers but
inhibited rAd5 F35 at low levels, suggesting Ad5 fiber was the
main neutralization target (Fig. 1C, left). In contrast, serum
from another subject neutralized both chimeras at high titers,
consistent with the presence of neutralizing antibodies directed
to both fiber and other capsid proteins (Fig. 1C, right).
Analysis of Ad5 neutralization in serum samples from the
VRC 006 trial subjects. Serum samples from 12 out of the 32
participants in VRC 006 were Ad5 seropositive at a titer of
?1:36 prior to vaccination, presumably due to prior natural
exposure to Ad5 virus. All but one serum sample from the 12
seropositive subjects inhibited transduction of the rAd35 F5
reporter, indicating the high prevalence of Ad5 fiber-related
neutralizing antibodies in these 12 subjects. Of these 12 serum
samples, 7 serum samples contained only Ad5 fiber-specific
neutralizing activity (58%) and 4 serum samples (33%) con-
tained neutralizing antibodies to both fiber and other capsid
proteins (Fig. 2, left and right, respectively). These seven se-
rum samples failed to block Ad35 entry, confirming their spec-
ificity for Ad5 (Fig. 2, left). This preliminary analysis revealed
two characteristic profiles of response, one in which neutraliz-
ing activity is focused mainly on Ad5 fiber and a second in
which recognition is directed to both fiber and other capsid
proteins. In the latter case, neutralization could also require
the concurrent presence of Ad5 fiber and other Ad5 capsid
proteins (Fig. 2, subject 1), suggesting either recognition of a
more complex conformational epitope or the synergistic effect
of low-level fiber and capsid specificities.
To characterize the nature of antibodies induced by vacci-
nation with rAd vectors in Ad5-naïve individuals, neutraliza-
tion profiles of these subjects were analyzed after a single
vaccination with the Ad5 vaccine. By 4 weeks after vaccination,
17 of the 20 Ad5-naïve subjects developed Ad5 neutralizing
antibodies at titers of ?1:36, while 3 subjects fell below this
threshold (Fig. 3A). All serum samples neutralized rAd5 F35,
indicating that capsid proteins other than fiber represented the
target of neutralization. Indeed, serum samples from 10 sub-
jects did not inhibit entry of the rAd35 F5 reporter, demon-
strating an absence of Ad5 fiber neutralizing activity in more
than half of the group. Serum samples that contained Ad5 fiber
neutralizing antibodies also had neutralizing antibodies to
other capsid proteins. Thus, Ad5-naïve subjects immunized
with Ad vaccines generated neutralizing antibodies to capsid
proteins other than fiber with the highest frequency.
Vaccination of individuals who were Ad5 seropositive elic-
ited antibodies that neutralized rAd5, rAd5 F35, and rAd35 F5
in all 12 subjects, thus showing high titer to both Ad5 fiber and
other capsid proteins (Fig. 3B). Because anti-fiber antibodies
were present in Ad5-seropositive subjects before vaccination,
immunization with rAd5 vectors increased anti-fiber titers and
expanded reactivity to other capsid proteins (Fig. 4, upper
versus lower right). Similarly, all seronegative subjects who
generated anti-Ad5 antibodies following vaccination generated
neutralizing antibodies to capsid proteins other than fiber,
while a subset also produced anti-fiber antibodies (Fig. 4, up-
per versus lower left).
The impact of neutralization activity on rAd vaccine immu-
nogenicity. To determine the impact of preexisting Ad5 neu-
tralizing activity on vaccine-induced immune responses, we
examined a larger number of subjects from trial HVTN 204, a
phase II study of the DNA/rAd Env A, B, and C; Gag; Pol; and
Nef vectors conducted with 180 individuals in North America.
Prior to vaccination, Ad5 seropositivity in trial participants was
55% (70 out of 127 samples analyzed). Fifty percent of the
seropositive subjects had neutralizing antibodies specific for
Ad5 fiber (Fig. 5A, left, Fiber). Antibodies to both fiber and
other capsid proteins were detected in 34% of subjects (Fig.
5A, left, Fiber?Capsid). Thus, similar to Ad5-seropositive
FIG. 2. Ad neutralizing titers in prevaccination serum samples from volunteers involved in the HIV vaccine trial VRC006 at week 0. Serum
samples from 32 volunteers were analyzed for seropositivity to Ad5, and 12 serum samples showed titers of greater than 36. These 12 serum samples
were used to neutralize Ad vectors, and their neutralizing titers to each vector were determined. Due to the limited amount of serum available,
the minimum titer was set at 1:36 and the maximum was 1:26,244. The serum samples can be grouped by their neutralizing activities into two major
groups: one group with neutralizing activity solely directed to Ad5 fiber (Fiber) and another group with neutralizing activity to fiber plus other Ad5
capsid proteins (Fiber?Capsid). In only serum sample number 1, neutralization required the concurrent presence of Ad5 fiber and other Ad5
VOL. 84, 2010 Ad5 IMMUNITY AND HIV-1 VACCINES633
by Jason Gall on January 15, 2010
VRC 006 volunteers, nearly every subject generated neutral-
izing antibodies to fiber, and neutralization targeted solely to
nonfiber capsid proteins was rare, ?4% (Fig. 5A, left, Capsid).
Serum samples from 11% of seropositive volunteers did not
neutralize either chimera, thus requiring the concurrent pres-
ence of capsid and fiber proteins for inhibition (Fig. 5A, left,
none versus Capsid and/or Fiber), similar to one subject from
VRC 006. Overall, the neutralization patterns were consistent
with the pattern of reactivity seen prior to vaccination in trial
VRC 006 (Fig. 4 and 5A). The Nab titers to Ad5 capsid and
fiber for each individual subject are also shown (Fig. 5A, right).
To determine the effect of these neutralizing antibodies on
vaccine-induced T-cell immunity, we analyzed ICS data that
were available for 121 subjects at week 28, 4 weeks after the
rAd HIV vaccine boost. Groups were stratified according to
the presence and pattern of rAd5 neutralizing antibodies. HIV
antigen-specific responses were first analyzed by magnitude in
seropositive and seronegative groups. The median CD4?and
CD8?Gag responses of Ad5 seronegative subjects (n ? 54) were
significantly higher than those of Ad5 seropositive subjects (n ?
67; P ? 0.001 and P ? 0.01, respectively) (Table 1). In contrast,
seropositive vaccinees remained similar (Table 1).
To analyze the effect of neutralizing antibodies directed to
different proteins of Ad5, the Ad5-seropositive group was split
into four subsets: Ad5 fiber only (fiber seropositive but capsid
seronegative, n ? 35), Ad5 fiber and capsid seropositive (n ?
22), Ad5 capsid only (n ? 3), and neutralizing target undefined
(n ? 7). The Ad5-capsid-only and the neutralizing-target-un-
defined subsets were too small for subset analysis. Ad5-fiber-
only seropositivity (n ? 35) correlated with reduced CD4?and
CD8?responses to Gag compared to Ad5 seronegativity
(CD4?P ? 0.004; CD8?P ? 0.04), and the responses did not
differ from those of the Ad5-seropositive group (Table 1; me-
FIG. 3. Adenoviral neutralizing antibody titers in serum samples from volunteers involved in the HIV vaccine trial VRC006 at 4 weeks
postvaccination. The postvaccination neutralizing titers are shown for naïve or seronegative subjects (A) and subjects with preexisting seropositivity
(B) (shown in the same order as shown in Fig. 2). For volunteers with preexisting seropositivity, all serum samples showed neutralizing antibodies
to both fiber and other capsid proteins, as they could neutralize rAd5, rAd5 F35, and rAd35 F5 but not rAd35 vectors. In naïve volunteers, two
neutralization patterns were seen following vaccination. One group could neutralize rAd5 and rAd5 F35 but not rAd35 or rAd35 F5, showing
capsid (non-fiber-mediated) neutralization; another group could neutralize rAd5, rAd5 F35, and rAd35 F5 but not rAd35, showing neutralization
directed to both fiber and other capsid proteins. Capsid denotes Ad5 capsid proteins other than fiber; Fiber?Capsid indicates Ad5 fiber and other
capsid proteins. The minimum titer was set at 1:36, and the maximum was 1:8,748.
634CHENG ET AL.J. VIROL.
by Jason Gall on January 15, 2010
dian CD4?response 0.013% versus 0.009%; median CD8?
response 0.007% versus 0.007%). Similarly, Ad5 fiber-plus-
capsid seropositivity (n ? 22) correlated with reduced CD4?
and CD8?responses compared to Ad5 seronegativity (Table 1;
CD4?P ? 0.002; CD8?P ? 0.02); and the responses did not
differ from those of the Ad5-seropositive group. Additionally,
there was no difference in Gag-specific responses between the
Ad5 fiber only and fiber-plus-capsid subsets (P ? 0.05). Thus,
the decline in Gag-specific T-cell response correlated signifi-
cantly with the presence of anti-fiber neutralizing antibodies,
and the presence of anti-capsid antibodies did not further
We then determined whether Ad5 fiber seropositivity af-
fected the response rate to Gag. Vaccine positive responders
were defined as showing significant responses after rAd5 vac-
cination by analysis of ICS data. In subjects with natural neu-
tralizing antibodies to Ad5 fiber, 12% (7 out of 57 rAd35
F5-seropositive subjects) showed positive CD4?T-cell re-
sponses to Gag (Fig. 5B). In individuals without Ad5 fiber
antibodies, 39% (25 out of 64 Ad35 F5-seronegative subjects)
had a positive CD4 T-cell response to Gag that was signifi-
cantly higher than the 12% response rate among seropositive
subjects (Fig. 5B, Gag, negative versus positive; P ? 0.001). On
the other hand, seropositivity to Ad5 fiber did not affect the
response to other antigens, such as EnvA (Fig. 5B, EnvA,
negative versus positive; P ? 0.05), for which the magnitude of
the response was also not affected. In individuals with anti-
capsid neutralizing antibodies, the frequency of positive CD4
responses to HIV Gag also trended lower compared to those
of capsid seronegative individuals, but it did not reach the
threshold P value of 0.001 required for statistical significance
because of the multiple group comparisons (Fig. 5C; P ? 0.02).
For example, 42% and 31% of the capsid seronegatives re-
sponded with positive CD4 responses to EnvA and Gag, re-
spectively, compared to 20% and 8% in seropositives. Similar,
but weaker, nonstatistically significant trends were observed
for other HIV antigens (data not shown). We also analyzed the
correlation between the titers of preexisting neutralizing anti-
bodies and the magnitude of responses generated by the vac-
cines. There were weak inverse relationships of antibody titer
to T-cell responses, but these correlations were not maintained
when relationships were restricted to only the people with
titers above the neutralization cutoff value. This analysis is
consistent with the data shown in the tables demonstrating that
the dominant effect was seropositivity and not the magnitude
of the neutralization titer. Thus, the data and analyses consis-
tently demonstrated that only seropositivity to Ad5 fiber was
significantly associated with a lower HIV Gag-specific T-cell
response compared to seronegativity.
The specificity and immune effects of the rAd5 neutralizing
antibodies of subjects receiving DNA/rAd vectors have been
analyzed in this study. We found that the molecular target of
neutralization differs depending on whether Ad5 immunity is
generated from natural infection or from vaccination with rep-
lication-defective viral vectors. Antibodies generated by natu-
ral infection are directed primarily to fiber components, while
vector exposure elicits responses primarily to capsid proteins
other than fiber. Injection of vector into Ad5 seropositive in-
dividuals elicits antibodies to both fiber and capsid in nearly all
subjects. The presence of antibodies to fiber in naturally in-
fected individuals reduces vaccine-induced immunity to HIV-1
Gag, whereas the response to Env is not substantially affected,
although the vaccines generated detectable immune responses
to both Gag and Env in Ad5 seronegatives. This observation
may be caused by differential trafficking and processing of an
aggregated protein in contrast to a transmembrane glycopro-
tein that could affect antigen presentation. It is also possible
that Ad5 immune effects are underestimated in this study be-
cause of the DNA immunization.
As shown in this study and documented elsewhere, Ad5
neutralizing antibodies are prevalent in humans. The sero-
prevalence seems to vary among populations from different
continents, and up to 85% of sera can be seropositive for Ad5
(13). The only study prior to the one reported here that had
assessed the contribution of individual capsid-specific antibod-
ies to viral neutralization in humans from natural infection
with Ad did not describe the frequency of samples positive for
FIG. 4. Distribution of adenoviral neutralization targets detectable
in serum samples from volunteers in the VRC006 trial. Data shown in
Fig. 2 and 3 are summarized based on the percentage of serum samples
containing antibodies directed to specific viral capsid proteins. In the
naïve volunteers, vaccination generated neutralization targeted mainly
to capsid proteins other than fiber alone. In the seropositive volun-
teers, neutralization directed to the fiber was detected broadly before
vaccination, whereas the neutralization extended to fiber plus other
capsid proteins after vaccination. Fiber and capsid are defined as
described in the legend for Fig. 3.
VOL. 84, 2010 Ad5 IMMUNITY AND HIV-1 VACCINES635
by Jason Gall on January 15, 2010
specific capsid proteins (18). Instead, a population median titer
was presented, which is an assessment of avidity, not fre-
quency. In addition, there was no statistical significance of the
apparent titer differences presented, and supporting data were
only from immunization of mice with rAd5. Taking the findings
together, it is unclear whether the median titers across the
populations of human samples could be extrapolated to deter-
mine immunodominant targets for neutralization. Thus, based
on the frequency assessment in the study presented here, neu-
tralizing antibodies to the fiber protein are more common than
antibodies to hexon. However, the frequency of hexon anti-
bodies was still significant and must be taken into account
when designing the next generation of rAd vaccine vectors.
Chimeric rAd5-based vectors have been demonstrated to
overcome vector neutralization (5, 18). Our results support the
use of chimeric Ads with non-Ad5 fibers in combination with
other non-Ad5 capsid proteins. Modification of capsid while
retaining an Ad5 fiber alone, as is done with mutant hyper-
variable region Ad5 vectors, is unlikely to fully overcome pre-
existing neutralizing antibodies from natural Ad infection. The
results also emphasize the contribution of anti-fiber antibodies
to neutralization generated by natural viral exposure, consis-
FIG. 5. Distribution of adenoviral neutralization targets detected in serum samples from volunteers in the HVTN204 trial at week 0 and its
association with the vaccine response rate. (A) Seventy serum samples from 127 volunteers showed preexisting neutralizing antibodies to Ad5 (left).
In the 70 positive serum samples, the percentage of sera that contained neutralizing antibodies directed to specific viral capsid proteins is shown
with a bar representing the 90% confidence interval for the true percentage of responders in each category. Fiber and capsid are defined as
described in the legend for Fig. 3. The distribution of preexisting anti-capsid (Ad5 F35) and anti-fiber (Ad35 F5) neutralizing antibody (Nab) titers
for the 121 volunteers who participated in HVTN204 clinical trials are also indicated (right). The cutoff titer (shown as a dashed line) for Nab
negatives was set at 36 for both anti-capsid (n ? 96) and anti-fiber (n ? 64) Nab. (B and C) The responders to the rAd5 HIV vaccine among 121
volunteers for whom valid immune response data are available were categorized according to Ad35 F5 seropositive or seronegative (positive or
negative Ad5 fiber neutralizing antibodies, respectively; panel B) and Ad5 F35 seropositive or seronegative (positive or negative Ad5 capsid
neutralizing antibodies, respectively; panel C). The percentage of responders with positive CD4?T-cell response to EnvA or Gag is shown, with
a bar representing the 90% confidence interval for the true percentage of responders in each category. ns, not significant, with P ? 0.001.
TABLE 1. Effect of Ad5 seropositivity on HIV vaccine vector-
Median response (% of cells positive
?P value compared to Ad5 seronegative?)
(n ? 54)
(n ? 67)
(n ? 35)
Ad5 fiber ?
(n ? 22)
0.013 (0.0009) 0.009 (0.004) 0.013 (0.002)
0.007 (0.007)0.007 (0.04)
0.021 (0.1)0.025 (0.3)
0.015 (0.07)0.021 (0.3)
0.016 (0.08) 0.024 (0.3)
0.007 (0.2)0.009 (0.4)
0.024 (0.1) 0.025 (0.4)
0.016 (0.2) 0.016 (0.4)
0.001 (0.5) 0.001 (0.8)
0.004 (0.053)0.005 (0.057) 0.002 (0.6)
0.013 (0.3)0.013 (0.4)
0.013 (0.6) 0.014 (0.9)
aThe magnitude of CD4?and CD8?T-cell responses to the indicated HIV
antigens in different serostatus groups.
636CHENG ET AL. J. VIROL.
by Jason Gall on January 15, 2010
tent with previous studies showing the importance of anti-fiber
antibody and its synergy with anti-penton antibody for neutral-
ization (7, 22). These antibodies are likely targeted to the fiber
knob (5). Since the modification of the hexon protein has been
shown to affect the distribution of cellular transduction in vivo
(20), it will be important to assess the anti-Ad and anti-trans-
gene immune responses to capsid-modified rAd vaccine vec-
Vaccination of naïve volunteers with rAd5 rarely generated
neutralizing antibodies targeted solely to fiber. This result may
reflect differences in the exposure to fiber and hexon in natural
infection compared to vaccination. During natural infection,
new fiber molecules are transcribed from viral genes in excess
of the amount of fiber incorporated into virions, and they may
be released from infected cells that undergo cytolysis. The
significant late protein synthesis, particularly of fiber, from
infected cells not only increases the fiber antigen content, but
also exerts biological effects on intercellular adhesion or sig-
naling through cellular receptors that will result in a very dif-
ferent set of immune and inflammatory responses than in
E1,E4-deleted vectors. In contrast, vaccination with the repli-
cation-incompetent rAd5 vector would result in exposure to
smaller amounts of fiber, as the viral proteins introduced in
vivo would be restricted to preformed virus, the incoming rep-
lication-incompetent virions of the E1,E4 replication defective
vector. An Ad virion is composed of 36 fiber protein monomers
and 720 hexon protein monomers (15), and vaccine recipients
would be expected to respond better to the more abundant
hexon protein. Viral replication in vivo may also affect the
presentation of epitopes to B cells and stimulation of helper
and memory T cells. Vaccination with rAd5 vectors in volun-
teers previously seropositive to Ad5 generated sera that tar-
geted all capsid components, including fiber, showing the mo-
bilization and recall of more diverse B-cell target repertoires to
fend off the vaccine vector.
Many studies have used intramuscular injection of rAd5
vectors to generate Ad5 immunity in an effort to mimic natural
exposure to infectious virus in animal models. Our study shows
that vaccination with rAd vectors generates a profile of neu-
tralizing antibodies different than natural infection, presum-
ably due to the differences of route, amount of viral exposure,
and capability of viral replication. Such differences call for
caution in interpreting animal model data from studies of
vector preexposure with respect to their effect on vaccine im-
munogenicity. In fact, these studies better reflect the effect of
repeated intramuscular administration of vector rather than
the effect of prior natural exposure.
While some data are available regarding the effect of pre-
existing neutralizing antibodies on the immunogenicity of
rAd5-based vectors (4, 14), the specificity of these antibodies
and correlation with immune stimulation has not been ana-
lyzed. In previous trials, there was a trend toward suppression
of immunity in the presence of preexisting Ad5 neutralizing
antibodies, especially for HIV-1 Gag-specific enzyme-linked
immunospot assay responses. Here, we show that such an effect
blunted both CD4?and CD8?T-cell Gag responses in sero-
positive volunteers, but we could not detect a significant effect
on Env responses after DNA prime/rAd5 boost vaccination. As
the response was not completely abolished in seropositive pop-
ulations (4, 14), it is likely that the effective dose of vector was
decreased but not completely inactivated. It is tempting to
suggest that efficient HIV Gag antigen presentation may need
a higher level or longer time of antigen expression than other
antigens. In support of this hypothesis, it has been shown that
the immunogenicity of HIV-1 Gag generated by DNA vacci-
nation can be more dependent on the formation of viral-like
particles than other antigens (1). The results shown here sug-
gest that rAd5-based vectors, especially Gag-encoding vectors,
are less likely to stimulate HIV-1 immunity in an Ad5 sero-
positive population; such a suppressive effect may also have
contributed to the lack of rAd5 vaccine efficacy in the recent
STEP trial. These data also document qualitative differences in
immunity between Ad5 infection and vaccination that may
affect the responses to subsequent rAd5 vector exposure. Pos-
sibly, these responses could also differentially affect the proin-
flammatory responses to vaccination that might affect suscep-
tibility to infection, a possibility raised in the STEP study.
These results also document a biological basis for differences
between responses to immunization with rAd5 vaccines in Ad5
naïve and seropositive subjects that are relevant to proposed
DNA/rAd5 efficacy studies; such studies should be preferably
performed in Ad5 seronegative subjects.
In summary, this study highlights differences in the specific-
ities of neutralizing antibodies generated by natural infection
compared to vaccination. Furthermore, we have shown that
anti-Gag cellular immune responses are more sensitive to the
effects of preexisting neutralizing antibody than those directed
against Env when using these vaccine formulations. The iden-
tification of the Ad5 vector proteins targeted by human neu-
tralizing antibodies also suggests that next generation chimeric
Ad vectors be developed without rAd5 hexon and fiber, which
could avoid preexisting immunity and increase the likelihood
of eliciting cellular immunity to HIV-1 antigens encoded in the
We thank Ati Tislerics for manuscript preparation, Brenda Hartman
for preparation of figures, and members of the Nabel lab for helpful
discussions and comments.
This research was supported by the Intramural Research Program of
the NIH, Vaccine Research Center, NIAID.
We declare that we have no material conflicts of interest, although
intellectual property applications have been filed through the National
Institutes of Health on the background DNA/rAd5 vaccine reported
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