A Novel HIV Vaccine Adjuvanted by IC31 Induces Robust
and Persistent Humoral and Cellular Immunity
Laura Pattacini1, Gregory J. Mize1, Jessica B. Graham1, Tayler R. Fluharty1, Tisha M. Graham1,
Karen Lingnau2, Benjamin Wizel2, Beatriz Perdiguero3, Mariano Esteban3, Giuseppe Pantaleo4,
Mingchao Shen1, Gregory A. Spies1, M. Juliana McElrath1,5, Jennifer M. Lund1,5*
1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2Intercell AG, Vienna, Austria, 3Centro
Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientı ´ficas, Madrid, Spain, 4Division of Immunology and Allergy, Department of Medicine and Swiss
Vaccine Research Institute, Lausanne University Hospital, Lausanne, Switzerland, 5Department of Global Health, University of Washington, Seattle, Washington, United
States of America
The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox
vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV
vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would
result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse
model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31H, and
the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31H facilitated
immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum
alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime
with IC31H and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector
memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is
promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune
Citation: Pattacini L, Mize GJ, Graham JB, Fluharty TR, Graham TM, et al. (2012) A Novel HIV Vaccine Adjuvanted by IC31 Induces Robust and Persistent Humoral
and Cellular Immunity. PLoS ONE 7(7): e42163. doi:10.1371/journal.pone.0042163
Editor: Cristian Apetrei, University of Pittsburgh Center for Vaccine Research, United States of America
Received May 18, 2012; Accepted July 2, 2012; Published July 25, 2012
Copyright: ? 2012 Pattacini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Bill & Melinda Gates Foundation (http://www.gatesfoundation.org) Collaboration for AIDS Vaccine Discovery [grant
numbers 38645 to MJM, 38599 to GP]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: K. Lingnau and B. Wizel are former employees of Intercell AG. This does not alter the authors’ adherence to all the PLoS ONE policies on
sharing data and materials.
* E-mail: firstname.lastname@example.org
Human immunodeficiency virus (HIV) is responsible for nearly
two million deaths annually, and although the overall incidence
appears to have stabilized, the epidemic continues to spread
(WHO, 2011). A vaccine represents the best possibility for
eradication of the virus, but despite unprecedented efforts, an
effective vaccine has not yet been developed.
Two recent vaccine efficacy trials provide clues to potential
components that can contribute to protective immunity against
HIV. Specifically, the Step study used a mixture of recombinant
adenovirus serotype 5 (Ad5) vectors expressing HIV-1 proteins
[1,2]. That study terminated early after interim analyses demon-
strated that the vaccine neither prevented infection nor lowered
viral load, and perhaps had the adverse effect of increasing HIV
acquisition in subjects with preexisting Ad5 neutralizing antibod-
ies. Though the reasons for this remain unclear, follow up
investigations indicated that adenovirus-specific CD4+ T cells
might have impacted the availability of potential HIV target cells
[3,4]. In contrast, the RV144 trial was the first ever to demonstrate
modest protection from HIV infection . That trial used an
ALVAC-HIV prime in combination with a VaxGen AIDSVAX
bivalent gp120 clade B/E protein boost. The regimen induced
mostly humoral and low-level CD4+ T-cell responses, supporting
the hypothesis that balancing both arms of the immune response
will induce improved protection.
New York vaccinia virus (NYVAC) vector is a highly attenuated
Copenhagen virus strain capable of inducing humoral and T-cell
responses [6,7]. NYVAC-CN54 encodes cell-released HIV-1 Env
gp120 and Gag/Pol/Nef, an intracellular polyprotein harboring
cytotoxic T lymphocyte epitopes . Preclinical studies in mice
demonstrated that this vector, when used as a boost after a DNA
prime, induces HIV-specific CD8+ T-cell responses and IgG
production [8,9]. In monkeys, a similar NYVAC vector expressing
HIV gp120 and SIV Gag/Pol/Nef induced CD4+ and CD8+ T
cells and antibodies to Env, with protection following SHIV89.6p
challenge . In a phase I clinical trial NYVAC-CN54 induced a
robust immune response, in particular Env-specific IFN-c
production by CD4+ and CD8+ T cells . Additionally,
vaccinia viruses are advantageous vaccine vectors because pre-
existing immunity at the population level is restricted to aged
groups, since smallpox vaccination was terminated in the mid-
1970s. Even in individuals with pre-existing immunity, smallpox-
specific T cells are less frequent in the mucosal tissues of healthy
PLoS ONE | www.plosone.org1July 2012 | Volume 7 | Issue 7 | e42163
volunteers than adenovirus-specific T cells , suggesting that
use of this vector may avoid problems encountered in the Step
study. Finally, vaccinia viruses are capable of activating innate
immune responses through TLR-dependent pathways [13,14].
TLR activation is recognized as a key component in several
vaccines, including the yellow fever vaccine, the activity of which
seems largely to be due to the generation of Th1-inducing mature
DCs . Such findings have opened a new field of research on
novel adjuvants, many of which are TLR ligands, to mimic the
pathogen-associated molecular patterns recognized during an
encounter with a natural pathogen.
Thus far, eleven TLRs have been identified in mice. Among
them, TLR3, TLR7, TLR8 and TLR9 recognize nucleic acids; in
particular, TLR9 recognizes CpG motif-containing DNA se-
quences. Several CpG oligodeoxynucleotide (CpG ODN) formu-
lations have already been used as adjuvants in vaccine studies for
infectious diseases, including HIV [16,17]. In this regard, CpG
ODN was shown to improve the humoral and cellular responses in
a vaccination regimen consisting of Gag protein prime and
adenovirus boost, both in mice  and in a primate model .
The recently developed compound IC31 consists of a combination
of ODN1a, a TLR9 ligand, and the antimicrobial peptide
KLKL5KLK, which contributes to the stabilization of ODN1a
and to depot formation . This combination was shown to
induce a strong cellular and humoral immune response through
activation of DCs and antigen-specific T-cell proliferation.
Furthermore, IC31 increases the activation and cytotoxic activity
of CD8+ T cells .
In this study we compared different vaccination regimens using
NYVAC-CN54 and recombinant Env-CN54 protein, in combi-
nation with alum alone, with IC31 alone, or with IC31 and alum
together. We evaluated induction of Env-specific antibodies,
CD4+ and CD8+ T-cell effector function, and the formation of
mucosal central and effector memory T-cell subsets. Our findings
indicate that IC31 greatly improves vaccination immunogenicity
in terms of the development of the Env-specific response.
Materials and Methods
Female BALB/c mice, 5 to 6 weeks old, were purchased from
Harlan Laboratory (Indianapolis, IN) and maintained in the Fred
Hutchinson Cancer Research Center (FHCRC) animal health
resource facility under pathogen-free conditions.
All experiments were approved by the FHCRC institutional
animal care and use committee. The Office of Laboratory Animal
Welfare has approved the FHCRC’s Animal Welfare Assurance
Purified recombinant HIV-1 Env-CN54 gp140 was manufac-
tured by Polymun (Vienna, Austria). IC31H is a proprietary
formulation developed by Intercell (Vienna, Austria) and was
supplied by the manufacturer. Alhydrogel (alum) was from
Brenntag (Frederikssund, Denmark). The development and
immunogenicity of NYVAC-CN54 were previously described
. Env peptides spanning the complete CN54gp140 protein
sequence were synthesized as 15-mers overlapping by 11 amino
acids by Biosynthesis (Lewisville, TX), and were pooled sequen-
tially at 40 peptides per pool (Table 1).
Vaccine regimens and injections
Mice were divided into 10 test cohorts of 15 mice each, except
for the two groups that received protein adjuvanted with alum
only, where nine mice each were used. Five cohorts received
NYVAC-CN54 as prime and CN54 protein as boost, while the
other five cohorts received CN54 protein as prime and NYVAC-
CN54 as boost. As a placebo control, two additional cohorts of
nine mice each received saline alone. For all test cohorts NYVAC-
CN54 was administered at 107PFU per injection; Env-CN54 was
given at 10 mg/mouse per injection. For Env-CN54 adjuvanted
with alum alone or with alum and IC31 together, protein was
mixed with alum (20 mg) for 1 hour at room temperature with
gentle agitation in a Tris buffered solution (20 mM Tris, 150 mM
NaCl, pH 7.5). Where indicated, IC31, at a concentration of
1.4 nmol ODN1a (‘‘IC31 medium’’) or 4 nmol ODN1a (‘‘IC31
high’’), was then added to the protein with alum. For protein
adjuvanted with IC31 alone, the same concentrations were used.
Mice were intramuscularly injected in the rear quadriceps using a
28-gauge insulin syringe. A schematic representation of the
injection schedules and regimens is shown in Figure 1.
Detection of HIV-1 Env-specific antibodies
Serum was obtained from mice at weeks 5, 11 and 15 and
ELISAs were performed following standard procedures. 2HB
plates (96-well; Nalge Nunc, Rochester, NY) were coated
overnight with 0.9 mg/ml HIV Env-CN54 protein, then washed
with buffer (150 mM NaCl, 0.1% Tween 20 in water), blocked for
1 h in 5% nonfat milk with 3% heat-inactivated goat serum and
0.2% Tween 20 in PBS, and then washed again. The serum
samples were serially diluted in blocking buffer, then added to the
wells in duplicate and incubated for 2 h at 37uC. The plates were
then washed, and HRP-conjugated anti-mouse IgG (Thermo
Scientific, Waltham, MA), anti-mouse IgG1-HRP, or anti-mouse
IgG2a-HRP (both from SouthernBiotech, Birmingham, AL) was
added at 1:2500 dilution and incubated for 1 h at 37uC. After
washing, the plates were developed with soluble 3,39-5,59-
tetramethylbenzene (BM blue POD substrate; Roche Applied
Science, Indianapolis, IN); the reaction was stopped by addition of
1 M H2SO4and the optical density (OD) was read at 450 nm
using a SpectraMax M2 plate reader (Molecular Devices,
Sunnyvale, CA). The reported titers correspond to the reciprocal
of the highest serum dilution showing a four-times higher OD
value than background.
Intracellular cytokine staining and measurement of
cellular proliferation by CFSE dilution
Fresh cells from individual spleens or MLN were stimulated for
5 h with one pool of Env-CN54 peptides, with 0.08% DMSO
(peptide diluent) alone as a negative control, or with 1 mg/ml of
Table 1. Aminoacid residues included in each of the four
peptide pools used for in vitro stimulations, and their location
in the Env protein structure.
Pool AA positionEnv structure
2 191–361 V3
4 507–665 gp41
IC31-Env and NYVAC-CN54 Immunogenicity
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anti-CD3 and 0.5 mg/ml anti-CD28 as a positive control, adding
10 mg/ml Brefeldin A (eBioscience, San Diego, CA) during the
incubation. The cells were then washed, blocked for 10 min with
0.5 mg/ml anti-CD16/CD32 in PBS containing 2% FBS and then
the surface staining was performed. The cells were then washed
and fixed with fixation permeabilization buffer (eBioscience) for
30 minutes at 4uC. The intracellular staining was then performed
and the samples were analyzed on a BD LSRII system (BD
Biosciences, Franklin Lakes, NJ). FACS data were analyzed using
FlowJo software (TreeStar, Ashland, OR). All antibodies used for
stimulation, sorting and staining were obtained from eBioscience,
and included anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7),
anti-CD127 (clone A7R34), anti-KLRG1 (clone 2F1), anti-CD62L
(clone MEL-14), anti-IFN-c (clone XMG1.2), anti-TNF-a (clone
MP6-XT22), anti-CD3 (clone 145-2C11), anti-CD28 (clone 37.51)
and anti-CD16/CD32 (clone 93).
To perform the cellular proliferation assay, 16106splenocytes
from each mouse were resuspended in PBS containing 0.1% BSA.
Carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Grand
Island, NY) was added to reach a final concentration of 1 mM, and
the cells were incubated at room temperature for 5 minutes.
Culture medium containing 10% FBS was then added and the
cells were incubated for 5 minutes on ice, washed twice and then
plated at a concentration of 105cells/well in the presence of Env-
CN54 peptide pool 1, DMSO alone, or anti-CD3/CD28 at the
same concentrations as used for the intracellular cytokine staining
stimulation. After three days of culture, cells were stained following
the intracellular cytokine staining protocol. Proliferation was
measured as the percentage of CFSE-low cells.
Data were analyzed by two-tailed unpaired Student’s t tests
using PRISM 5 software (GraphPad Software, La Jolla, CA).
Welch’s correction was applied for non-equally distributed
All vaccinations elicited a humoral response to Env
We first examined the Env-specific antibody response following
administration of various vaccine regimens (Fig. 1). After priming
with protein, the groups receiving Env-IC31 induced at least a
three log increase in their IgG2a titer at week 5 as compared to
Env-alum alone (Fig. 2A). This difference was statistically
significant when the high dose of IC31 plus alum was used,
where a seven log-difference was achieved (p,0.05). We observed
a similar trend for total IgG and IgG1. After boosting with
NYVAC-CN54, serum IgG2a titers at week 11 trended slightly
higher in the groups that were primed with protein adjuvanted
with IC31 plus alum as compared to protein with either adjuvant
alone, although the differences were not significant. At week 15 we
did not observe any significant differences between the groups
Among mice primed with NYVAC-CN54 and boosted with
protein plus adjuvant, there were no differences in antibody
induction at weeks 11 or 15, regardless of adjuvant (Fig. 2B).
Figure 1. Schematic representation of the vaccination protocol. Three to five mice for each timepoint were injected at weeks 0 and 3 with
the prime and at weeks 6 and 9 with the boost. Mice were either injected with saline (negative control, three mice per group), with protein plus alum
alone, with protein plus a medium or high dose of IC31 alone, with protein plus alum and a medium or high dose of IC31, or with NYVAC-CN54 (five
mice per each group per timepoint). At week 5, 11 or 15, a subset of mice were sacrificed and antibody and T-cell responses were analyzed ex vivo.
IC31-Env and NYVAC-CN54 Immunogenicity
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Env-specific CD4+ T-cell responses are increased in
magnitude and durability by IC31-protein prime/NYVAC-
Next we examined CD4+ T-cell responses two weeks after
protein prime (week 5) and two and six weeks after boosting with
NYVAC-CN54 (Fig. 1). We used decreased expression of CD62L
to indicate general T-cell activation, and observed a significant
increase in CD4+ T-cell activation at week 5 when the medium
dose of IC31 alone or the high dose of IC31 plus alum was used in
combination with protein versus the saline control (p,0.05 each;
Fig. 3A). Although the differences were not significant in all
groups, there was also a general trend for increased CD4+ T-cell
activation following the NYVAC-CN54 boost versus the saline
control at weeks 11 and 15 (Fig. 3A).
We also evaluated CD4+ T-cell function by measuring cytokine
secretion upon in vitro stimulation. Priming with Env-CN54
adjuvanted with IC31 at any dose, with or without alum, induced
a higher percentage of cytokine producing cells at week 5 as
compared to the saline control or to priming with Env-CN54
adjuvanted with alum alone (Fig. 3B). The median frequency of
cytokine-producing CD4+ T cells detected after stimulation with
peptide pool 1, which induced the majority of detectable
responses, ranged from 0.023% when the adjuvant was the
medium dose of IC31 alone, to 0.072% with the high dose of IC31
plus alum. Boosting with NYVAC-CN54 induced a two- to three-
fold increase in the CD4+ T-cell response in all regimens.
Moreover, mice immunized with the high dose of IC31 plus alum
had a significantly higher frequency of cytokine-producing CD4+
T cells versus those receiving the medium dose plus alum or alum
alone (0.204% for IC31 high/alum vs. 0.062% for IC31 medium/
alum and 0.018% for alum alone, p=0.016 and p=0.033,
respectively; Fig. 3B). Six weeks after the final immunization (week
15), we again observed that inclusion of IC31 with the protein
prime induced a CD4+ T-cell response of greater magnitude than
inclusion of alum alone (Fig. 3B). The CD4+ T-cell response to
peptide pool 1 is shown longitudinally in Fig. 3C, demonstrating
that adjuvanting the protein prime with the high dose of IC31,
alone or with alum, induced the greatest expansion of Env-specific
CD4+ T cells and this population was still present at week 15.
When the regimens were reversed, with NYVAC-CN54 as
prime and the protein-adjuvant combinations as boost, we
Figure 2. Robust antibody responses are induced by all vaccination regimens. Serum levels of total IgG (green triangles), IgG1 (black
circles) and IgG2a (red circles) were measured at weeks 5, 11 and 15. A) Mice were primed at weeks 0 and 3 with Env-CN54 gp140 with alum alone, or
a medium (‘‘med’’) or high concentration of IC31 with or without alum, and boosted at week 6 and 9 with NYVAC-CN54. B) Mice were primed at
weeks 0 and 3 with NYVAC-CN54 and boosted at week 6 and 9 with Env-CN54 gp140 protein in combination with the adjuvants as indicated. Total
IgG could not be detected in the control mice. The graphs show the antibody titers detected by ELISA at weeks 5, 11, and 15. Bars correspond to the
IC31-Env and NYVAC-CN54 Immunogenicity
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IC31-Env and NYVAC-CN54 Immunogenicity
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observed a general trend for increased T-cell activation when the
adjuvant included IC31 (Fig. 3D). CD4+ T-cell responses were of
lower magnitude at week 11 as compared to protein prime/
NYVAC-CN54 boost. We could still detect a slightly superior
adjuvant activity with the high dose of IC31 plus alum, although
this response was only significantly different from alum alone and
the medium dose of IC31 plus alum (p,0.05 each; Fig. 3E). At
week 15, we detected a very low percentage of Env-specific CD4+
T cells secreting cytokines, and there were no significant
differences between groups (Fig. 3E).
Protein/IC31 adjuvant prime, NYVAC-CN54 boost
regimens induce superior Env-specific CD8+ T-cell
We next evaluated the effect of the different regimens on the
activation status and cytokine production of CD8+ T cells. As a
marker of CD8+ T-cell activation we again utilized decreased
expression of CD62L. Two weeks after priming with protein/
adjuvant, there was a trend toward an increase in the percentage
of activated CD8+ T cells for all vaccine groups versus the saline
control (Fig. 4A). Two weeks after NYVAC-CN54 boost, we
detected a significant increase in CD8+ T-cell activation in all
vaccine groups versus the saline control (p,0.01 for alum only and
IC31 medium plus alum, p,0.05 for all others; Fig. 4A). At week
15, although the trend was the same, a significant difference versus
saline was observed only in the groups primed with protein and
alum alone or protein with the medium dose of IC31 alone
(p,0.05 each; Fig. 4A).
We next examined the effect of the different regimens on CD8+
T-cell cytokine production. We did not detect significant antigen-
specific CD8+ T-cell responses in any group following the protein/
adjuvant primes (Fig. 4B). After boosting, we detected cytokine
secretion in all groups, although there were significant differences
in frequencies between groups (Fig. 4B), with the greatest
difference observed between protein prime with the high dose of
IC31 plus alum versus protein prime with alum alone (2.74%
median frequency of response to peptide pool 1 versus 0.5%;
Fig. 4C). At week 15, we also observed a robust Env-specific
effector response in the CD8+ T-cell compartment in all groups
primed with protein and IC31, while the group primed with
protein and alum alone had a significantly lower response rate
(p,0.05 each; Fig. 4B). In summary, priming with protein and the
high dose of IC31 plus alum generated an Env-specific CD8+ T-
cell response with the highest magnitude and durability (Fig. 4C).
When the regimens were reversed, with NYVAC-CN54 prime
and the protein-adjuvant combinations as boost, CD8+ T-cell
activation followed a trend similar to that observed with the
protein prime/NYVAC-CN54 boost regimens (Fig. 4D). Follow-
ing protein boost, the groups for which IC31 was used as an
adjuvant showed an increase in CD8+ T-cell activation as
compared to alum alone or the saline control. At the memory
timepoint, we observed a trend toward increased CD8+ T-cell
activation in the groups boosted with protein and IC31 compared
to protein with alum alone or the saline control, although this only
reached significance for the high dose of IC31 alone versus the
saline control (p,0.05; Fig. 4D). While all adjuvant/protein
formulations elicited an elevated frequency of CD8+ T-cell
response over the saline control, none reached statistical signifi-
cance either directly after boosting or at the memory timepoint
IC31 promotes an enhanced memory response
Since priming with protein adjuvanted with IC31 induced
CD4+ and CD8+ T-cell responses that persisted, we further
investigated the ability of this regimen to induce immunological
memory. We first determined the number of memory precursors
two weeks after NYVAC-CN54 boost by measuring the number of
antigen-specific CD8+ T cells (producing TNF-a and/or IFN-c
following stimulationwith peptide
CD127+KLRG1- . Mice primed with protein adjuvanted
with the high dose of IC31, with or without alum, had significantly
more memory precursors (p,0.05 each vs. alum only, Fig. 5A),
demonstrating that IC31 is superior to alum alone in terms of
inducing CD8+ T-cell memory precursors.
We next determined the number of central memory (TCM) and
effector memory (TEM) cells six weeks after boost by distinguishing
Env-specific CD8+ T cells by cytokine expression and further
gating on CD62L positive or negative cells, respectively . The
greatest expansion of the central memory cell compartment
occurred when mice were primed with protein adjuvanted with
the high dose of IC31 plus alum (Fig. 5B). In all cases, adjuvanting
with IC31 resulted in significantly higher expansion than with
alum alone (p,0.05 each; Fig. 5B). Additionally, CD8+ T cells at
the memory timepoint had significantly increased proliferative
capacity when the high dose of IC31 was used, with or without
alum (p,0.01 each vs. alum only; Fig. 5C). This is consistent with
previous studies demonstrating that TCMcells are superior to TEM
cells in proliferation [23,24]. Finally, we found that a protein
prime including IC31, with or without alum, resulted in
significantly increased effector memory cell generation (p,0.05
each vs. alum only; Fig. 5D).
pool1) that were
Env-specific CD8+ T cells are induced in the mesenteric
lymph nodes when the protein prime includes IC31
We next examined the Env-specific CD8+ T-cell response in the
mesenteric lymph nodes (MLN) following the protein prime/
NYVAC-CN54 boost vaccination regimens. Both immediately
following NYVAC-CN54 boost and at a memory timepoint there
were cytokine-secreting CD8+ T cells present in the MLN,
regardless of the adjuvant used. The percentages were similar at
the two timepoints and the pattern was comparable to that
observed in the spleens (Fig. 6A–B). Specifically, at week 11, the
high dose of IC31 plus alum induced the highest percentage of
Env-specific CD8+ T cells (0.094%, compared to 0.065% for IC31
Figure3.DurableEnv-CN54specificCD4+ +T-cellresponses aredetectedfollowingvaccinationwhenEnv/alum/IC31isusedasaprime.
alum and then boosted with NYVAC-CN54 at three week intervals. A) Frequencies of activated CD4+ T cells as indicated by low CD62L expression at
weeks 5, 11 and 15. Each data point corresponds to a single mouse in the experimental group. B) Percentage of CD4 T cells secreting cytokines (TNF-a
and IFN-c) upon stimulation with four peptide pools covering the full length of Env-CN54 gp140, as determined by ICS. C) Kinetic analysis of the
frequencies of cytokine secreting CD4+ T cells following vaccination. Data points correspond to the medians of the frequencies obtained by stimulation
with peptide pool 1. D–E) Mice were primed with NYVAC-CN54 and boosted with Env-CN54 gp140 in combination with adjuvants as listed. (D)
Frequencies of activated CD4+ T cells as indicated by low CD62L expression at weeks 11 and 15. Each data point corresponds to a single mouse in the
experimental group. (E) Percentage of CD4+ T cells secreting cytokines at weeks 11 and 15. For B and E, data points following stimulation with peptide
pool1 (redcircles),pool2 (greensquares),pool3 (greentriangles)andpool4(blacktriangles)aredistinguished,whilethedottedlinecorrespondstothe
average obtained upon DMSO stimulation. Bars correspond to median values (medians for peptide pool 1 stimulation only in B and E). *p,0.05.
IC31-Env and NYVAC-CN54 Immunogenicity
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IC31-Env and NYVAC-CN54 Immunogenicity
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high alone, 0.058% for IC31 medium alone, 0.036% for IC31
medium plus alum and 0.052% for alum alone). Six weeks after
boosting with NYVAC-CN54 we detected Env-specific TEMin the
MLN of mice that received the protein prime adjuvanted with the
high dose of IC31 plus alum. The number of TEMin these MLN
was significantly higher than in mice primed with protein and
alum alone, where Env-specific TEMwere virtually undetectable
(p,0.05; Fig. 6C). We could not detect Env-specific CD4+ T cells
in the MLN of mice from any group (data not shown), despite
detecting a potent response in the spleen (Fig. 3).
In our study, we tested the novel adjuvant IC31 in combination
with recombinant Env-CN54 protein in a heterologous prime-
boost vaccination regimen with a NYVAC-CN54 vector encoding
for the same Env variant. To our knowledge, this is the first study
Figure 4. Durable Env-CN54 gp140 specific CD8+ + T-cell activation and cytokine secretion are detected following vaccination. (A–C)
Mice were primed with Env-CN54 gp140 in combination with alum alone, or with a medium (‘‘med’’) or high concentration of IC31 with or without
alum, and then boosted with NYVAC-CN54 at three week intervals. A) Frequencies of activated CD8+ T cells as indicated by low CD62L expression at
weeks 5, 11 and 15. Each data point corresponds to a single mouse in the experimental group. B) Percentage of CD8+ T cells secreting cytokines upon
stimulation with the four peptide pools, as determined by ICS. C) Kinetic analysis of the frequencies of cytokine secreting CD8+ T cells following
vaccination. Data points correspond to the medians of the frequencies obtained by peptide pool 1 stimulation. D–E) Mice were primed with NYVAC-
CN54 and boosted with Env-CN54 gp140 in combination with adjuvants as indicated. D) Frequencies of activated CD8+ T cells as indicated by low
CD62L expression at weeks 11 and 15. Each data point corresponds to a single mouse in the experimental group. E) Percentage of CD8+ T cells
secreting cytokines at weeks 11 and 15. Labels are as shown in Figure 3. *p,0.05; **p,0.01.
Figure 5. The memory T-cell pool is enhanced by Env/IC31 prime. A) At week 11, the numbers of CD8+ memory precursor cells (IFN-c+ and/
or TNF-a+ CD8+ KLRG1-low CD127+) in the spleen were determined for mice that were primed with Env-CN54 gp140 with various adjuvants as
indicated and boosted with NYVAC-CN54. The graph shows the results obtained after in vitro stimulation with peptide pool 1. B) At week 15, memory
T-cell numbers were assessed in the spleen. Central memory T cells were defined as CD8+ T cells secreting IFN-c and/or TNF-a with high expression of
CD62L. The graph depicts central memory cell numbers induced by the various vaccination regimens following in vitro re-stimulation with peptide
pool 1. C) Proliferation of spleen CD8+ T cells from week 15 was determined by CFSE dilution. The graph shows the frequency of proliferating CD8+ T
cells following in vitro stimulation with peptide pool 1 for 3 days. D) Effector memory T cells from week 15 were defined as CD8+ T cells secreting IFN-
c and/or TNF-a, expressing low levels of CD62L. The graph shows the values obtained after in vitro stimulation with peptide pool 1. Bars indicate the
median value. *p,0.05 and **p,0.01. Each data point corresponds to a single mouse in the experimental group.
IC31-Env and NYVAC-CN54 Immunogenicity
PLoS ONE | www.plosone.org8 July 2012 | Volume 7 | Issue 7 | e42163
utilizing IC31 for an HIV vaccine immunogenicity study. IC31
was previously shown to induce Th1-like antibody immunity and
to facilitate isotype switching to IgG2a [25,26]. In the context of
HIV vaccines, this characteristic is particularly valuable, since the
importance of antibody functional activities other than neutrali-
zation, such as ADCC, has recently been highlighted by the
correlates analysis of the RV144 trial . In our study, we
observed that adjuvanting a protein prime with IC31 induced
increased levels of IgG2a and lowered the ratio of IgG1/IgG2a as
compared to priming with protein plus alum alone. We also
showed that NYVAC-CN54 prime followed by protein boost
induced an elevated level of IgG2a antibodies, regardless of the
adjuvant used (Fig. 2), confirming that NYVAC-CN54 is an
excellent prime for antibody induction, as previously reported .
Isotype switching to IgG2a has been shown to be dependent on
production of IFN-c . While CD4+ T-cell activation was
increased following protein prime at weeks 5 and 11 regardless of
the adjuvant used, inclusion of IC31 maintained this activation at
a later timepoint (Fig. 3A). Induction of a CD4+ T-cell response is
essential to prevent the exhaustion of CD8+ memory T cells upon
re-challenge . This is particularly important when considering
the vaccination protocols often used for HIV vaccine trials, which
utilize multiple challenges with the same antigen, thus potentially
leading to exhaustion of CD8+ T cells in the absence of antigen-
specific CD4+ T cells. We hypothesize that the inclusion of IC31
prevented this exhaustion by maintaining CD4+ T-cell activation
(Fig. 4B). Importantly, the responses that we detected for both
CD4+ and CD8+ T cells were mostly induced by Env peptide pool
1, which includes sequences from the V1/V2 loops. Antibody
responses to the same region of similar Env proteins have been
recognized as one of the principal correlates of risk, associated with
a 43% reduction in infection in the RV144 trial vaccine recipients
Finally, the inclusion of IC31 in a protein prime induced a
higher number of Env-specific CD8+ T cells in the MLN (Fig. 6),
while we did not detect any antigen-specific CD4+ T-cell response
at this site. This is likely to be advantageous for an HIV vaccine,
since it will not increase the frequency of activated CD4+ T cells,
which are potential targets of the virus, at an important site of
replication. Moreover, the induction of CD8+ T-cell memory at a
mucosal site, and specifically in the gut, is likely to be important in
preventing HIV replication early after infection. This effect on
mucosal immunity might be one of the causes for the protection
from Chlamydia infection in a recent vaccination study where IC31
was used as the adjuvant .
Our results can likely be explained by the capacity of IC31 to
signal through TLR9/MyD88 and induce dendritic cell matura-
tion, which is thought to then be responsible for driving Th1
responses . The different cellular distribution of TLR9 in mice
and humans has historically posed a problem with results gained
from mouse models not translating into humans . Importantly,
however, IC31 has been administered in two clinical trials of
Figure 6. Detection of Env-CN54 gp140 specific CD8+ + T cells in the GALT of vaccinated mice. The frequencies of CD8+ T cells secreting
cytokines in response to CN54 peptide pools were evaluated in the mesenteric lymph nodes (MLN) of vaccinated mice at week 11 (A) and at week 15
(B). Graphs show the median CD8+ T-cell responses from the MLN (white bars) and from spleen (black bars) following stimulation with peptide pool
1. C) Effector memory T-cell numbers within the MLN were determined as described for Figure 5. Each data point corresponds to a single mouse in
the experimental group.
IC31-Env and NYVAC-CN54 Immunogenicity
PLoS ONE | www.plosone.org9 July 2012 | Volume 7 | Issue 7 | e42163
tuberculosis vaccines in naı ¨ve as well as in BCG-vaccinated and
TB-positive subjects, where it was shown to induce a Th1 immune
response with IFN-c secretion by PBMC . Thus, we expect
IC31 to have a similar potent effect when included in an HIV
vaccine administered to humans.
Our study also demonstrates the advantages of combining two
distinct adjuvants, namely IC31 and alum, for inducing both T-
cell and antibody responses. IC31 has been shown to induce strong
type 1 immune response , while alum is known to induce type
2 immune responses .
In this vaccination schedule, the order of NYVAC-CN54 and
protein is important for the induction of a strong cellular response.
The efficacy of NYVAC-CN54 as a boost was reported in a recent
NHP study . Our study likewise found that NYVAC-CN54
provided an excellent boost in terms of CD4+ and CD8+ T-cell
activation and function.
Our study reveals multiple innovative findings for the field of
HIV vaccinology. First, we showed that IC31 used in combination
with Env-CN54 in the priming phase of a vaccination regimen
induces isotype switching and the production of Env-specific
IgG2a. IC31 also increases the magnitude of CD4+ and CD8+
Env-specific T-cell responses, the development of a central
memory and effector memory compartment and the presence
and persistence of antigen-specific T cells in gut-associated
lymphoid tissues. Second, we demonstrated that priming with
NYVAC-CN54 followed by protein/adjuvant boost does not
induce B and T-cell immunity to the same extent as the reverse
vaccination strategy. Taken together, our study supports the use of
IC31 as a protein adjuvant for future HIV vaccine trials, in
combination with a viral vector boost, such as NYVAC-CN54.
We thank Stephen Voght for critical reading of the manuscript. We thank
the James B. Pendleton Charitable Trust for their generous equipment
Conceived and designed the experiments: LP GJM MS GAS MJM JML.
Performed the experiments: LP GJM JBG TRF TMG. Analyzed the data:
LP JML. Contributed reagents/materials/analysis tools: KL BW BP ME
GP MJM. Wrote the paper: LP GJM MS GAS MJM JML.
1. Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, et al. (2008)
Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study):
a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet
2. McElrath MJ, De Rosa SC, Moodie Z, Dubey S, Kierstead L, et al. (2008) HIV-
1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort
analysis. Lancet 372: 1894–1905.
3. Frahm N, DeCamp AC, Friedrich DP, Carter DK, Defawe OD, et al. (2012)
Human adenovirus-specific T cells modulate HIV-specific T cell responses to an
Ad5-vectored HIV-1 vaccine. J Clin Invest 122: 359–367.
4. Perreau M, Pantaleo G, Kremer EJ (2008) Activation of a dendritic cell-T cell
axis by Ad5 immune complexes creates an improved environment for replication
of HIV in T cells. J Exp Med 205: 2717–2725.
5. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, et al.
(2009) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in
Thailand. N Engl J Med 361: 2209–2220.
6. Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, et al. (1992)
NYVAC: a highly attenuated strain of vaccinia virus. Virology 188: 217–232.
7. Gomez CE, Najera JL, Krupa M, Perdiguero B, Esteban M (2011) MVA and
NYVAC as vaccines against emergent infectious diseases and cancer. Curr Gene
Ther 11: 189–217.
8. Gomez CE, Najera JL, Jimenez EP, Jimenez V, Wagner R, et al. (2007) Head-
to-head comparison on the immunogenicity of two HIV/AIDS vaccine
candidates based on the attenuated poxvirus strains MVA and NYVAC co-
expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-
Nef proteins of clade B. Vaccine 25: 2863–2885.
9. Wild J, Bieler K, Kostler J, Frachette MJ, Jeffs S, et al. (2009) Preclinical
evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC
vaccines in the Balb/C mouse model. Viral Immunol 22: 309–319.
10. Mooij P, Balla-Jhagjhoorsingh SS, Beenhakker N, van Haaften P, Baak I, et al.
(2009) Comparison of human and rhesus macaque T-cell responses elicited by
boosting with NYVAC encoding human immunodeficiency virus type 1 clade C
immunogens. J Virol 83: 5881–5889.
11. Harari A, Bart PA, Stohr W, Tapia G, Garcia M, et al. (2008) An HIV-1 clade C
DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional,
and long-lasting T cell responses. J Exp Med 205: 63–77.
12. Perreau M, Welles HC, Harari A, Hall O, Martin R, et al. (2011) DNA/
NYVAC vaccine regimen induces HIV-specific CD4 and CD8 T-cell responses
in intestinal mucosa. J Virol 85: 9854–9862.
13. Zhu J, Martinez J, Huang X, Yang Y (2007) Innate immunity against vaccinia
virus is mediated by TLR2 and requires TLR-independent production of IFN-
beta. Blood 109: 619–625.
14. Delaloye J, Roger T, Steiner-Tardivel QG, Le Roy D, Knaup Reymond M, et
al. (2009) Innate immune sensing of modified vaccinia virus Ankara (MVA) is
mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome. PLoS
Pathog 5: e1000480.
15. Querec T, Bennouna S, Alkan S, Laouar Y, Gorden K, et al. (2006) Yellow fever
vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to
stimulate polyvalent immunity. J Exp Med 203: 413–424.
16. Krieg AM (2007) Antiinfective applications of toll-like receptor 9 agonists. Proc
Am Thorac Soc 4: 289–294.
17. Kwant A, Rosenthal KL (2004) Intravaginal immunization with viral subunit
protein plus CpG oligodeoxynucleotides induces protective immunity against
HSV-2. Vaccine 22: 3098–3104.
18. Tritel M, Stoddard AM, Flynn BJ, Darrah PA, Wu CY, et al. (2003) Prime-boost
vaccination with HIV-1 Gag protein and cytosine phosphate guanosine
oligodeoxynucleotide, followed by adenovirus, induces sustained and robust
humoral and cellular immune responses. J Immunol 171: 2538–2547.
19. Wille-Reece U, Flynn BJ, Lore K, Koup RA, Miles AP, et al. (2006) Toll-like
receptor agonists influence the magnitude and quality of memory T cell
responses after prime-boost immunization in nonhuman primates. J Exp Med
20. Fritz JH, Brunner S, Birnstiel ML, Buschle M, Gabain A, et al. (2004) The
artificial antimicrobial peptide KLKLLLLLKLK induces predominantly a
TH2-type immune response to co-injected antigens. Vaccine 22: 3274–3284.
21. Schellack C, Prinz K, Egyed A, Fritz JH, Wittmann B, et al. (2006) IC31, a novel
adjuvant signaling via TLR9, induces potent cellular and humoral immune
responses. Vaccine 24: 5461–5472.
22. Cui W, Kaech SM (2010) Generation of effector CD8+ T cells and their
conversion to memory T cells. Immunol Rev 236: 151–166.
23. Kaech SM, Hemby S, Kersh E, Ahmed R (2002) Molecular and functional
profiling of memory CD8 T cell differentiation. Cell 111: 837–851.
24. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, et al. (2003)
Lineage relationship and protective immunity of memory CD8 T cell subsets.
Nat Immunol 4: 225–234.
25. Riedl K, Riedl R, von Gabain A, Nagy E, Lingnau K (2008) The novel adjuvant
IC31 strongly improves influenza vaccine-specific cellular and humoral immune
responses in young adult and aged mice. Vaccine 26: 3461–3468.
26. Bernardo L, Pavon A, Hermida L, Gil L, Valdes I, et al. (2011) The two
component adjuvant IC31(R) potentiates the protective immunity induced by a
dengue 2 recombinant fusion protein in mice. Vaccine 29: 4256–4263.
27. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, et al.
(2012) Immune-correlates analysis of an HIV-1 vaccine efficacy trial.
N Engl J Med 366: 1275–1286.
28. Stoecklinger A, Eticha TD, Mesdaghi M, Kissenpfennig A, Malissen B, et al.
(2011) Langerin+ dermal dendritic cells are critical for CD8+ T cell activation
and IgH gamma-1 class switching in response to gene gun vaccines. J Immunol
29. West EE, Youngblood B, Tan WG, Jin HT, Araki K, et al. (2011) Tight
regulation of memory CD8(+) T cells limits their effectiveness during sustained
high viral load. Immunity 35: 285–298.
30. Cheng C, Cruz-Fisher MI, Tifrea D, Pal S, Wizel B, et al. (2011) Induction of
protection in mice against a respiratory challenge by a vaccine formulated with
the Chlamydia major outer membrane protein adjuvanted with IC31(R).
Vaccine 29: 2437–2443.
31. Rehli M (2002) Of mice and men: species variations of Toll-like receptor
expression. Trends Immunol 23: 375–378.
32. van Dissel JT, Soonawala D, Joosten SA, Prins C, Arend SM, et al. (2011)
Ag85B-ESAT-6 adjuvanted with IC31(R) promotes strong and long-lived
IC31-Env and NYVAC-CN54 Immunogenicity
PLoS ONE | www.plosone.org10 July 2012 | Volume 7 | Issue 7 | e42163
Mycobacterium tuberculosis specific T cell responses in volunteers with previous Download full-text
BCG vaccination or tuberculosis infection. Vaccine 29: 2100–2109.
33. Lindblad EB (2004) Aluminium compounds for use in vaccines. Immunol Cell
Biol 82: 497–505.
34. Flynn BJ, Kastenmuller K, Wille-Reece U, Tomaras GD, Alam M, et al. (2011)
Immunization with HIV Gag targeted to dendritic cells followed by recombinant
New York vaccinia virus induces robust T-cell immunity in nonhuman primates.
Proc Natl Acad Sci U S A 108: 7131–7136.
IC31-Env and NYVAC-CN54 Immunogenicity
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