JOURNAL OF VIROLOGY, Jan. 2009, p. 540–551
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 2
Human Immunodeficiency Virus Type 1 Env Trimer Immunization of
Macaques and Impact of Priming with Viral Vector or Stabilized
Andreas Mo ¨rner,1Iyadh Douagi,1Mattias N. E. Forsell,1,2Christopher Sundling,1Pia Dosenovic,1
Sijy O’Dell,2Barna Dey,2Peter D. Kwong,2Gerald Voss,3Rigmor Thorstensson,1John R. Mascola,2
Richard T. Wyatt,2and Gunilla B. Karlsson Hedestam1*
Swedish Institute for Infectious Disease Control, 171 82 Solna, and Department of Microbiology, Tumor and Cell Biology,
Karolinska Institutet, 171 77 Stockholm, Sweden1; Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922; and
GlaxoSmithKline Biologicals, Rixensart, Belgium3
Received 26 May 2008/Accepted 31 October 2008
Currently there is limited information about the quality of immune responses elicited by candidate human
immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we
describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus
macaques by three selected immunization regimens. We used the previously described YU2-based gp140
protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus
replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound
conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression
platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively
lymphocytes recognizing common determinants between the core and trimers to broaden the immune response.
The results presented here demonstrate that the platform by which Env trimers were delivered in the priming
(either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with
stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of
HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained
in animals immunized with trimers alone and was shown to be mediated by CD4?T cells. However, this
regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further
immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing
determinants of HIV-1 Env.
The human immunodeficiency virus type 1 (HIV-1) enve-
lope glycoprotein (Env) consists of a trimer of noncovalently
associated gp120/gp41 heterodimers, which form the func-
tional viral spike and mediate entry into CD4 and CCR5 re-
ceptor-positive host target cells. The exterior envelope glyco-
protein, gp120, and the transmembrane glycoprotein, gp41, are
the sole virally encoded targets for neutralizing antibodies on
the surface of the virus and likely represent a critical immu-
nogenic component for an effective prophylactic vaccine
against HIV-1 (7, 23, 33). The HIV-1 Envs are also potential
targets for cell-mediated immune responses, and as such, their
inclusion in future HIV-1 vaccine candidates may contribute to
the induction of both protective antibody and cellular immune
responses (25). In attempts to elicit antibodies that recognize
the functional Env spike, soluble trimeric molecules containing
full-length gp120 covalently linked to the gp41 ectodomain
have been designed (5, 24, 40, 46, 53, 54). An incremental
advance in neutralizing antibody elicitation using soluble tri-
meric Env spike mimetics compared to the use of monomeric
gp120 was observed (28, 55), but further improvements in
trimer immunogen design are still needed both to mimic better
the functional viral spike and to elicit broadly neutralizing
antibodies (reviewed in reference 7 and 37).
The immunogenicity of cleavage-defective Env trimers derived
from the primary R5 isolate YU2, possessing heterologous tri-
merization motifs derived either from T4 bacteriophage (foldon)
or from the transcription factor GCN4, were examined in several
small animals studies (6, 15, 28, 55). However, to date these
trimeric Env immunogens were not analyzed for their ability to
elicit neutralizing antibodies and Env-specific T-cell responses in
nonhuman primates. Other oligomeric Env proteins, such as the
SF162 gp140 proteins with or without a deletion of the second
major variable region (?V2), were evaluated with nonhuman
that gp140SF162?V2 administered in the MF59 adjuvant medi-
ated protection against mucosal challenge with the SHIV-162P4
virus (2), implicating Env-directed immune responses in mediat-
ing protection against this homologous virus challenge.
The capacity of different Env immunogens to stimulate hu-
moral and cellular responses was also evaluated using genetic
means of expression, such as plasmid DNA or recombinant
viral vectors, followed by immunization of purified Env protein
in an adjuvant to boost antibody responses (15, 34, 45, 51).
* Corresponding author. Mailing address: Department of Microbiol-
ogy, Tumor and Cell Biology, Karolinska Institutet, Box 280, S-171 77
Stockholm, Sweden. Phone: 46-8-457-2568. Fax: 46-8-337272. E-mail:
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 12 November 2008.
While such heterologous immunization regimens may enhance
Env-directed cellular immune responses, little is known about
the quality of neutralizing antibody responses induced by viral
vector priming followed by a protein boost or about the rela-
tive responses elicited by regimens consisting of purified Env
protein in an adjuvant using homologous or heterologous pro-
tein priming/boosting. One potential concern when Env is ex-
pressed in vivo from DNA or viral vectors is that the actual
dose and the antigenic integrity of the immunogen are not
easily assessed. For example, incorrectly folded but immuno-
genic Env protein released from dying cells in vivo may ad-
versely affect the quality of the elicited antibody response.
Since many candidate vaccines which are currently moving into
clinical trials rely on in vivo genetic expression (9, 10, 18, 38,
39, 48, 49), analysis of the quality of antibody responses elicited
by genetic platforms is warranted and is an aim of our present
Previously we performed a head-to-head study using rabbits to
characterize neutralizing antibody responses stimulated by se-
quential administrations of YU2 Env protein trimers emulsified
in the Ribi adjuvant compared to two immunizations of single-
round infectious recombinant Semliki Forest virus (rSFV) parti-
cles expressing YU2 trimers followed by a boost with YU2 trimeric
proteins in an adjuvant (15). SFV is an alphavirus, genetically
related to Sindbis virus and Venezuelan equine encephalitis virus
(VEE), two other viruses for which single-round replicon systems
have been developed (8, 41). SFV has been used extensively in
preclinical immunogenicity experiments (3, 4, 14, 15, 16, 19, 21,
29, 30, 32, 47), human vaccine candidates based on VEE are
already in clinical trials (11), and currently the chimeric VEErep/
SINenv vector is one of the most frequently used alphavirus-
based vector systems for preclinical HIV-1 vaccine studies (35,
51). In our previous study, we demonstrated that rSFV infection
of BHK-21 cells in vitro resulted in the secretion of homogeneous
and stable gp140 trimers into the culture medium (15). Here, to
evaluate recombinant alphavirus priming in greater detail, we
performed a comprehensive study with nonhuman primates to
examine both antibody and cellular responses induced by gp140
trimers with and without rSFV priming. We inoculated cynomol-
gus macaques with Env trimers administered as purified protein
in the AS01B adjuvant system from GlaxoSmithKline Biologicals
(GSK) or expressed in vivo from rSFV particles. We used cleav-
age-defective (?) gp140 trimers possessing the heterologous
foldon (F) trimerization motif (gp140-F) (54), hereafter referred
to as gp140-F trimers. As an additional arm of the study, we used
a modified gp120 core protein in AS01B as a prime, followed by
test the concept of “immunofocusing” by first inoculating with the
stable gp120 core, followed by the gp140-F proteins in sequence
to focus responses on the shared and conserved core elements of
the two forms of Env. The modified gp120 core proteins, hereaf-
ter referred to as “stable core,” contain pocket-filling mutations
and were stabilized by disulfide-linked cysteine pairs spanning the
inner and outer domains of gp120, as previously described (56).
These modifications were designed to reduce the flexibility of
gp120 and to improve presentation of conserved but discontinu-
ous cross-domain antigenic surfaces of Env, such as the CD4-
binding site. We hypothesized that priming with a stable core
protein and boosting with gp140-F trimers would favor the ex-
pansion of Env-specific B cells recognizing common determinants
between the two immunogens and that this might translate into
an increased breadth of neutralization. We also wished to deter-
mine if priming with stable cores and boosting with trimers would
alter the Env-specific T-cell response compared to immunization
with gp140-F trimers alone, since this may represent a strategy to
focus the cellular response on conserved sequences common to
the two immunogens. Our results show that the properties of the
Env antigen used to prime the response are more important for
shaping the overall Env-specific immune response than the plat-
form by which Env is delivered. Priming with the stable core
protein and boosting with trimers strikingly focused the T-cell
response on core sequences of Env even as measured after the
trimer boost. However, this regimen did not improve the neutral-
izing antibody response, suggesting that further immunogen de-
sign efforts are required to successfully focus the B-cell response
on conserved structural determinants on the functional viral spike
to stimulate antibodies possessing an increased breadth of neu-
MATERIALS AND METHODS
Cells, recombinant viruses, and antibodies. BHK-21 cells (ATCC) were cul-
tured in Glasgow minimal essential medium (MEM) (Invitrogen, Carlsbad, Cal-
ifornia), supplemented with 5% fetal calf serum, 10 mM HEPES, 10% tryptose
phosphate, 2 mM L-glutamine, penicillin, and streptomycin (Sigma, St. Louis,
MO). Sequences encoding YU2gp140(?/FT), flanked by XmaI/XhoI, were
amplified by PCR from YU2gp140FT/pCDNA3.1(?), respectively (54). The
sequences were inserted into pSFV10 (22) to generate rSFV-gp140-F trimer
particles. Single-round infectious recombinant pSFV-gp140-F particles (rSFV-
gp140-F) were produced by cotransfecting BHK-21 cells with in vitro-transcribed
vector and split-helper mRNA (3) according to the standard protocol, and
titration of viral stocks was performed using BHK-21 cells as described previ-
ously (22). The monoclonal antibodies (MAbs) used in this study were 17b, F105,
and 447-52D, generously obtained from James Robinson, Marshal Posner, and
Susan Zolla-Pazner, respectively, and D7324, an antibody directed against the C5
region of gp120, purchased from Aalto Bio Reagents Ltd. (Dublin, Ireland).
Expression and purification of Env immunogens. The gp140-F trimers were
produced by transient transfection of adherent 293F cells or Freestyle 293F
suspension cells (Invitrogen). Adherent cells were transfected in Dulbecco’s
modified Eagle medium–10% heat-inactivated fetal bovine serum–0.1 mM MEM
nonessential amino acid solution (Invitrogen) media, using LipofectAMINE
2000 (Invitrogen) as per the manufacturer’s instructions. One day posttransfec-
tion, serum-containing Dulbecco’s modified Eagle medium was replaced with
serum-free 293 SFM II medium (Invitrogen). Beginning 2 days after transfection,
cell culture supernatants were collected daily and fresh 293 SFM II medium
added to the cell culture flasks until the sixth day posttransfection. Suspension
cells were transfected in Gibco Freestyle293 expression medium by using the
293Fectin transfection reagent in Opti-MEM according to the manufacturer’s
instructions (Invitrogen). The supernatant was collected 4 days after transfec-
tion. Following collection, all supernatants were centrifuged at 3,500 ? g, filtered
through a 0.22-?m filter, and supplemented with Complete, EDTA-free protease
inhibitor cocktail (Roche) and penicillin-streptomycin (Invitrogen) for storage at
4°C until further purification. The highly glycosylated and His6-tag-containing
YU2gp140-F trimers were purified from sterile-filtered, serum-free medium in a
three-step process. First, the protein was captured via the N-linked glycans with
lentil-lectin affinity chromatography (GE Healthcare, Uppsala, Sweden). After
extensive washing in phosphate-buffered saline (PBS), the protein was eluted
with PBS–1 M ?-?-mannopyranoside–0.5 M NaCl–10 mM imidazole, subse-
quently captured in the second step via the His tag by nickel chelation chroma-
tography (GE Healthcare), and then washed and eluted with a 300 mM imida-
zole-containing PBS buffer. Finally, the YU2gp140-F trimers were separated
from lower-molecular-weight proteins by gel filtration chromatography using a
Superdex 200 26/60 prep-grade column and the A ¨KTA Fast protein liquid chro-
matography system (GE Healthcare). The stable core proteins used in the cur-
rent study are a slightly modified version of the cysteine-stabilized Ds12F123
protein described previously (56). Following analysis of the core?V3 structure
(20), the Ds12F123 core protein was redesigned to enhance protein folding by
the addition of 13 residues at the base of the V3 loop, with the original GAG
tripeptide substitution of the V3 region replaced with 16 residues, RPNNGGS
VOL. 83, 2009 IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES541
GSGGNMRQA (Ds12F123V3S; B. Dey et al., unpublished data). The
Ds12F123V3S stable core protein was produced by transient transfection of
adherent 293F cells in serum-free medium as described above. Sterile-filtered
supernatant containing the stable core proteins was applied to an immunoglob-
ulin G (IgG) 17b affinity column. After extensive washing with PBS, core protein
was eluted from the column with 100 mM glycine–Tris HCl–150 mM NaCl, pH
2.8, immediately neutralized with Tris base, pH 8.5, and then dialyzed against
PBS–0.5 NaCl, pH 7.4. All proteins were spin concentrated with Amicon Ultra
30,000-molecular-weight-cutoff centrifugal filter devices (Millipore, Bedford,
MA) to a concentration between 1 and 3 mg/ml.
Biochemical analysis of Env immunogens. Purified proteins were analyzed for
conformational integrity and oligomeric status prior to inoculation. Conforma-
tional integrity was confirmed by immunoprecipitating purified proteins with two
conformation-sensitive MAbs: 17b (coreceptor site directed) and F105 (CD4bs
directed), as well as with 447-52D V3-directed antibody. Briefly, 5 ?g of protein
was coincubated for 1 h at room temperature (RT) with 15 ?g antibody and 30
?l Protein A beads (GE Healthcare) in 500 ?l PBS. After extensive washing
three times with PBS–0.5 M NaCl followed by one wash with PBS, beads with
bound antibody-antigen complexes were heated to 100°C for 5 min in 1?
NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen), supplemented with 1?
NuPAGE sample reducing agent (Invitrogen), and subjected to gel electrophore-
sis. The proteins were resolved on a NuPAGE 4 to 12% bis-Tris gel. The
oligomeric status of the purified proteins was analyzed by resolving 10 ?g protein
on a NuPAGE 4 to 12% bis-Tris gel under “blue native” conditions (40). Briefly,
samples diluted in 2? sample buffer (100 mM Tris HCl, 100 mM morpholinepro-
panesulfonic acid, 40% glycerol, 0.1% Serva-G, pH 7.7) were analyzed on a
NuPAGE 4 to 12% bis-Tris gel (Invitrogen) at 4°C at 50 V for 18 h. The running
buffer contained 50 mM Tris-HCl, 50 mM morpholinepropanesulfonic acid, pH
7.7, and to the cathode, 10 mg Serva-G (Serva Electrophoresis GmbH, Heidel-
berg, Germany) per 500 ml running buffer was added. Thyroglobulin and ferritin
(GE Healthcare) were used as molecular weight markers.
Animals and inoculations. Sixteen female cynomolgus macaques (Macaca
fascicularis) of Chinese origin, 5 to 6 years old, were housed in the Astrid
Fagraeus laboratory at the Swedish Institute for Infectious Disease Control.
Housing and care procedures were in compliance with the provisions and general
guidelines of the Swedish Animal Welfare Agency. All procedures were ap-
proved by the Local Ethical Committee on Animal Experiments. Animals were
housed in pairs in 4-m3cages, enriched to allow expression of physiological and
behavioral needs. They were habituated to the housing conditions for more than
6 weeks before the start of the experiment and subjected to positive-reinforce-
ment training to reduce the stress associated with experimental procedures. All
immunizations and blood sampling were performed under sedation with ke-
tamine (10 mg/kg of body weight) given intramuscularly (Ketaminol, 100 mg/ml;
Intervet, Sweden). Macaques were weighed and examined for swelling of lymph
nodes and spleen at each immunization or sampling occasion. Before entering
the study, all animals were confirmed to be negative for simian immunodefi-
ciency virus, simian T-cell lymphotropic virus, and simian retrovirus type D.
The 16 cynomolgus macaques (3 groups of 5 animals and 1 naive control animal)
were inoculated five times with the immunogens described above (Table 1). Immu-
nizations were performed at weeks 0, 3, 8, 12, and 18 by intramuscular injection,
except for rSFV-gp140-F, which was given subcutaneously. All protein immuniza-
tions were administered in combination with the AS01B adjuvant system from GSK.
Protein doses were 200 ?g per animal for the first inoculation and 100 ?g for the
following injections. The SFV-gp140-F dose used in the first two immunizations in
group 3 was 5 ? 108IU per injection. Vaccines were given in a total volume of 1 ml,
divided equally between the left and right hind legs. Blood samples were taken
before and 2 weeks after each immunization. As a negative control, one animal was
immunized five times with the AS01B adjuvant system alone using the same immu-
nization interval as described above.
ELISA. HIV-1 gp120-specific serum IgG was measured by enzyme-linked
immunosorbent assay (ELISA). Briefly, Nunc Maxisorp microtiter plates were
coated with insect-cell-produced YU2 gp120 protein at 1 ?g/ml in 50 mM PBS
overnight (ON) at ?4°C. After blocking in PBS containing 2% milk, serum
samples were added to and incubated for 2 h at RT. gp120-specific IgG was
detected by adding secondary biotinylated anti-monkey IgG antibody (Jackson
ImmunoReserach, Suffolk, United Kingdom) and streptavidin conjugated to
horseradish peroxidase (Mabtech, Stockholm, Sweden) followed by O-phenyl-
enediamine (Sigma, Schnelldorf, Germany). Between each incubation step, the
plates were washed six times with PBS supplemented with 0.05% Tween 20.
Substrate reactions were terminated with 2.5 M H2SO4, and the optical density
(OD) was read at 492 and 650 nm. The OD-at-50-nm (OD50) titers for each
sample were calculated by interpolating from the mean OD50value calculated
from controls using the formula [(ODmax? ODmin)/2] ? ODmin. When antibody
responses against denatured epitopes were investigated, gp120 was denatured by
boiling for 2 min in 1% sodium dodecyl sulfate and 50 mM dithiothreitol (Sigma)
and then diluted and analyzed as described above. As controls in this assay, we
used two monoclonal antibodies, F105, which recognizes a conformation-sensi-
tive epitope in gp120, and an anti-C5 antibody, D7324, which recognizes a linear
epitope in gp120, to confirm that the boiling resulted in a loss of conformational
but not linear epitopes. The assay with binding antibodies against gp140-F or the
stable core protein measured in serum samples after the second and fourth
immunizations were performed as described above with the following modifica-
tions: specific IgG was detected with horseradish peroxidase-conjugated second-
ary anti-human IgG (Fc region) (Jackson Laboratories) using 3,3?,5,5?-tetram-
ethylbenzidine (Bio-Rad) as a substrate. The reaction was stopped by adding 1 M
H2SO4, and the OD was read at 450 nm.
Virus neutralization assays. Sera from immunized animals were tested for
virus neutralization capacity against a panel of diverse HIV-1 isolates. Neutral-
ization assays were performed using a single-round-infection HIV-1 Env
pseudovirus assay and TZM-bl target cells as described previously (27, 45). Env
pseudoviruses were prepared by cotransfecting 293T cells with an Env expression
plasmid containing a full gp160 env gene and an env-deficient HIV-1 backbone
vector (pSG3?Env). To determine the serum dilution that resulted in a 50%
reduction in relative luminescence units, serial dilutions of sera were performed
and the neutralization dose-response curves were fitted by nonlinear regression
using a four-parameter-hill slope equation programmed into the JMP statistical
software program (JMP 5.1; SAS Institute Inc., Cary, NC). The results are
reported as the serum neutralization ID50, which is the reciprocal of the serum
dilution producing 50% virus neutralization. Peptide competition neutralization
assays were done in the same assay format as the neutralization assay, except that
the control or test peptide was added to serum 30 min prior to the addition of
virus. The V3 peptide sequences used in this study were synthesized by SynPep
(Dublin, CA) and were based on the YU2 sequence (TRPNNNTRKSINIGPG
RALYTTG). A scrambled V3 peptide (IGPGRATRPNNNFYTTGTRKSIH)
was used as a negative control.
Diverse HIV-1 virus isolates, including viruses from clades A, B, and C, were
used in the neutralization assays. Clade B viruses included a panel of Env
pseudoviruses that were recently characterized and recommended for use in
assessing neutralization by HIV-1 immune sera (26). Several investigators also
provided replication-competent viruses or functional Env plasmids for pseudovi-
ruses. Dana Gabuzda (Dana Farber Cancer Institute) provided the Env plasmids
for YU2 and murine leukemia virus. Env plasmids for SF162 and JRFL were
provided by Leonidas Stamatatos (Seattle Biomedical Research Institute) and
James Binley (Torrey Pines Institute), respectively. The clade A DJ263.8 se-
TABLE 1. Immunizationsa
Content of immunization no. (wk):
1 (0)2 (3) 3 (8)4 (12) 5 (18)
E71, E72, E75, E76, E87
E89, E90, E91, E95, E96
E73, E74, E77, E78, E88
aAll protein immunizations (gp140-F and stable core; groups 1 to 3) and all control immunizations (group 4) were given intramuscularly with the AS01B adjuvant
system. SFV-gp140-F immunizations (group 2) were given subcutaneously. Animals were bled 2 weeks after each immunization. For details, see Materials and Methods.
bn, no. of animals.
542MO ¨RNER ET AL. J. VIROL.
quence was cloned from the original peripheral blood mononuclear cell
(PBMC)-derived virus provided by Francine McCutchan and Vicky Polonis (U.S.
Military HIV Research Program), and the clade C MW965 Env plasmid was
obtained from the AIDS Research and Reagent Repository. The BaL.01 Env
(45) and SS1196.1 Env (26) plasmids were recently described by our laboratory.
Peptides and cells for enzyme-linked immunospot (ELISPOT) assay. Peptide
pools, comprised of 15-mer peptides overlapping by 10 residues, were purchased
(New England Peptide LLC, Gardner, MA). Two different peptide pools based
on the YU2 gp140 sequence were used: “gp140-F core” covered the conserved
regions of gp120, corresponding to the stable core immunogen, and “gp140-F
noncore” covered the remaining parts of YU2 gp140. A peptide pool based on
the HXBc2 stable core immunogen sequence was also used. PBMC were isolated
from EDTA blood by Ficoll-Paque PLUS (GE Healthcare Biosciences AB,
Uppsala, Sweden) separation, frozen in R20 medium with 10% dimethylsulfox-
ide and stored at ?150°C or in liquid nitrogen.
For the ELISPOT assays, MSIPN4550 plates (Millipore, Bedford, MA) were
prewetted with 40 ?l 35% ethanol, washed six times with distilled H2O, and
coated with the anti-gamma interferon (IFN-?) MAb GZ-4 (Mabtech, Nacka,
Sweden) at 10 ?g/ml in PBS ON at 4°C. The plates were then blocked with 150
?l R10 medium for 1 h at 37°C. The blocking solution was removed without
washing, and monkey PBMC, thawed and rested ON in R10 medium at 37°C
with 5% CO2, were added at 2 ? 105cells/well in duplicates with medium alone,
phytohemagglutinin (5 ?g/ml), or one of three overlapping peptide pools at 2.5
?g/ml. The cell viability was always ?90%. After 20 h of incubation in 5% CO2
at 37°C, biotinylated anti-IFN-? MAb 7-B6-1 (Mabtech) was added at 1 ?g/ml
and the plates were incubated for 2 h at RT followed by 1 h of incubation at RT
with streptavidin-alkaline phosphatase (Mabtech). Then 5-bromo-4-chloro-3-in-
dolylphosphate–nitroblue tetrazolium substrate (Sigma-Aldrich, St Louis, MO)
was added, and spots were allowed to develop for 30 min before the enzymatic
reaction was stopped. Volumes were 100 ?l/well, and the plates were washed six
times with PBS between all steps except where indicated. The spots were counted
using an ImmunoSpot series 4 analyzer (CTL, Aalen, Germany) and expressed
as numbers of spot-forming cells per 106PBMC. For experiments with CD8?
T-cell-depleted PBMC, CD8?cells were removed by magnetic cell sorting using
a nonhuman primate-specific CD8 depletion kit (Miltenyi Biotec Inc., Auburn,
CA). The purity of the CD8?-depleted fraction was determined by flow cytom-
etry after staining with anti-CD3-APC-Cy7 (clone SP34-2), anti-CD4-AmCyan
(clone L200), and anti-CD8-Pacific Blue (clone RPA-T8) (all from BD Bio-
sciences, San Jose, CA).
Biochemical characterization of Env immunogens and study
design. A schematic of the two Env proteins used in this study is
trimers and stable Ds12F123V3S core proteins used for immuni-
zations in this study, we immunoprecipitated the two protein
immunogens with three MAbs: 17b, which binds a determinant
overlapping the gp120 coreceptor binding site; F105, which binds
to a surface overlapping the CD4 binding site; and 447-52D,
which recognizes the V3 region of many isolates possessing a
GPGR motif at the tip of their V3 region (including YU2 and
HXBc2). Free proteins and protein-antibody complexes were
separated by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis and visualized by Coomassie blue staining. As expected,
stable core protein was precipitated with 17b but not with 447-
52D or F105, consistent with the protein lacking essential 447-
52D binding elements in the V3 loop (17) and with previously
published data on gp120 proteins harboring Phe 43 cavity-filling
mutations (50, 56) (Fig. 1B). Analysis by Blue Native gel electro-
phoresis confirmed that the mobility of the majority of the
gp140-F protein was consistent with that of a trimer while the
mobility of the stable core protein was consistent with the size of
FIG. 1. Design and biochemical characterization of HIV-1 envelope glycoproteins. (A) Linear (sequence) and tertiary (structure) schematic
representation of antigens used for immunization. Trimers of gp140-F were based on YU2 and include the full-length gp120 sequence with N and
C termini (N and C), variable regions (V1/V2/V3/V4 and V5), and the gp41 ectodomain (gray). They are cleavage defective (the REKR sequence
at the cleavage site was changed to SEKS to maintain a covalent gp120-gp41 association) and contain a heterologous foldon trimerization motif
(F) (yellow). Stable cores were based on HXBc2 and lack N and C termini, the V1/V2/V3 regions, and gp41. They were modified by structure-based
design to contain pocket-filling mutations (M95W, T257S, A433M, and S375W, which eliminates F105 recognition) (13, 56) and extra cysteine
bonds between amino acids 96 and 275 and amino acids 109 and 428, as indicated by dotted lines in the linear schematic and green bars in the
tertiary cartoon; these disulfides stabilize the CD4-bound conformation of gp120 (56). The inner domain of the HXBc2 core is colored blue and
the outer domain red. Numbering of amino acids is based on the HXBc2 numbering convention. (B) Purified proteins were analyzed for their
antigenic profiles by immunoprecipitation with the three monoclonal antibodies: 17b (coreceptor site directed), F105 (CD4bs directed), and
447-52D (V3 directed). The eluted proteins were resolved on a NuPAGE 4-to-12% bis-Tris gel (left panel). (C) The oligomeric status of the
proteins was analyzed by blue native gel electrophoresis. Thyroglobulin and ferritin (GE Healthcare) were used as molecular mass markers.
VOL. 83, 2009 IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES543
a loop-deleted gp120 monomer (Fig. 1C). Expression of gp140
trimers from the rSFV particles was analyzed as described previ-
The design for the present immunogenicity study involving
16 female cynomolgus macaques is outlined in Table 1. Five
monkeys (E71, E72, E75, E76, and E87) were inoculated five
times with purified gp140-F trimers combined with the GSK
AS01B adjuvant system (group 1). Five monkeys (E89, E90,
E91, E95, and E96) were inoculated twice with 5 ? 108IU
rSFV particles expressing gp140-F trimers, followed by three
immunizations with purified gp140-F trimers/adjuvant (group
2). A final five monkeys (E73, E74, E77, E78, and E88) were
immunized twice with the stable core proteins followed by
three immunizations with gp140-F trimers/adjuvant (group 3).
As a negative control for the neutralization assay, one animal
(E93) was inoculated with the AS01B adjuvant alone. The
interval between inoculations was 1 month, and the animals
were bled 2 weeks after each immunization for isolation of
PBMC and collection of sera.
Analysis of sera from Env-immunized macaques. Levels of
HIV-1 Env-binding antibodies were measured by standard
ELISA using YU2 gp120-coated wells, and OD50titers were
calculated as described in Materials and Methods. All animals
in group 1 mounted high antibody responses after a single Env
immunization, consistent with the potent immunogenicity pro-
vided by the combination of Env and the AS01B adjuvant
system (Fig. 2, top left panel). The Env-specific binding re-
sponse was boosted by the second immunization, after which
the antibody response plateaued. In group 2, Env-specific an-
tibody responses were also detectable after the first rSFV-
gp140-F immunization, but these responses were considerably
lower than group 1 titers (Fig. 2, top right panel). A second
rSFV-gp140-F immunization did not boost binding antibody
levels, possibly due to the impact of antivector immune re-
sponses as previously reported (31, 47). However, antibody
responses were readily boosted by one subsequent immuniza-
tion with gp140-F trimer protein and reached levels similar to
those in group 1. The stable core protein used for priming in
group 3 elicited lower levels of gp120-binding antibodies than
did the trimers (Fig. 2, bottom left panel), despite the same
amount of protein being administered and use of the same
adjuvant. However, the responses increased after the first
boost with gp140-F trimer proteins, and after the second boost
with the protein trimers, the responses reached a plateau sim-
ilar to that observed in the other groups. The geometric mean
ELISA titers for the three groups are shown in the lower right
panel of the figure. There was no difference in the preimmune
reactivity to Env among the animals in the three groups (see
Fig S1A in the supplemental material), and an intra-assay
control run on all ELISA plates confirmed that all assays were
equally sensitive (see Fig S1B in the supplemental material).
Full titration curves of all individual animals are presented in
Fig S1C in the supplemental material.
FIG. 2. Env binding antibodies in serum from immunized animals. The titers of Env-binding antibodies were measured in sera 2 weeks after
each immunization using a standard ELISA with gp120-coated wells. The OD50titer for each sample was calculated by interpolating from the mean
OD50value calculated from controls: [(ODmax? ODmin)/2] ? ODmin. The results for individual animals, as well as group means, are shown.
Preimmune Env-directed reactivity in sera diluted 100-fold was also determined and was shown not to differ between the groups (see Fig. S1A in
the supplemental material). “T” indicates gp140-F trimer protein immunization, “S” indicates SFV-gp140-F immunization, and “C” indicates
stable core protein immunization.
544 MO ¨RNER ET AL.J. VIROL.
Neutralizing antibody responses in Env-immunized macaques.
We next measured neutralizing activity in sera collected after
the second, fourth, and fifth immunizations. We used a panel
of recombinant viruses pseudotyped with the “tier 1” Env
glycoproteins MN, HXBc2, SF162, BaL, SS1196 (clade B), and
two non-clade B viruses, MW965 (clade C) and DJ263 (clade
A) and the “tier 2” Env glycoproteins YU2, JRFL, TRO.11,
and 6535.3. The data are presented as 50% neutralization titers
(50% inhibitory dilution [ID50]) with the serum ID50neutral-
izing titer of 20 highlighted as a dotted line (Fig. 3). The most
potent neutralizing activity in the post-2 sera was displayed by
animals immunized with the gp140-F trimer protein adminis-
tered in the AS01B adjuvant (group 1), where relatively high
neutralization titers against HXBc2, MN, SF162 (Fig. 3), and
the clade C virus MW965 (see Fig S2 in the supplemental
material) were elicited in all animals. Weaker or variable neu-
tralizing activity was observed against BaL, YU2, and SS1196,
and no neutralization was observed against the tier 2-type
viruses, JRFL, 6535.3, and TRO.11 (Fig. 3 and data not shown;
see also Fig S2 in the supplemental material).
FIG. 3. Neutralizing antibody responses against clade B viruses. Serum samples from each animal taken 2 weeks after the second, fourth, and
fifth immunizations were analyzed for neutralizing activity using the TZM-bl/Luc neutralization assay and the following Env glycoproteins: MN,
HXBc2, SF162, BaL.01, SS1196.01, JRFL, and YU2 (clade B). Serial serum dilutions from individual monkeys were tested, and the data are
presented as 50% neutralization titers (ID50). Neutralizing ID50titers above 20 are considered positive, as indicated by the dotted lines. “T” refers
to gp140-F trimer protein immunization, “S” refers to SFV-gp140-F immunization, and “C” refers to stable core protein immunization.
VOL. 83, 2009 IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES545
In animals primed with rSFV-expressing gp140-F trimers
and boosted with the gp140-F trimer protein in an adjuvant,
there was no consistent neutralizing activity after the first two
immunizations. After two booster immunizations with the
gp140-F protein, there was neutralizing activity against MN,
HXBc2, and SF162 in most animals, but the activity against all
viruses except MN was generally lower than in group 1. Over-
all, rSFV priming did not confer an advantage to the neutral-
izing response elicited by the gp140-F trimeric protein alone.
Similarly, there was no detectable neutralizing activity against
any of the viruses after two inoculations of the stable core
protein in an adjuvant (group 3, post-2). After two booster
immunizations with the gp140-F protein in an adjuvant (group
3, post-4), consistent neutralizing activity was observed only
against MN and SF162. There was a tendency toward more
consistent neutralization of BaL and SS1196 in the post-5
serum from group 3 than in group 1, suggesting a possible
advantage with the stable core-prime, trimer-boost regimen.
High neutralization titers against the clade C virus, MW965,
was observed in the post-4 and post-5 sera from all three
groups, while no or very weak neutralization was detected
against the tier 2-type viruses, JRFL, 6535, and TRO.11, for
most sera (Fig. 3 and data not shown; see also Fig S2 in the
supplemental material). Serum from the naive control animal
(group 4) tested negative for neutralization of all HIV-1 vi-
ruses in the assay, and all samples from groups 1 to 4 tested
negative against a murine leukemia virus pseudotype control,
confirming that the assay was specific for HIV-1 Env-directed
neutralization (data not shown).
Mapping specificities of anti-Env serum antibody responses.
To better understand what antibody specificities mediated the
neutralizing responses against MN, SF162, HXBc2, and
MW965, we performed peptide inhibition assays using the
post-5 serum from each hyperimmune animal. The results
from these experiments are shown as percent reduction of the
ID50neutralization values, where only values greater than a
50% reduction in neutralizing activity were considered reli-
able. Values in excess of a 50% reduction in ID50neutraliza-
tion are shown in Fig. 4. By using a YU2-derived V3 peptide
and a scrambled control V3-derived peptide, we found that
V3-specific antibodies were responsible for a significant frac-
tion (up to 90% in some sera) of the neutralizing activity
against SF162 and MN, and this was similar in all three groups
(Fig. 4, top and lower panel). It was previously shown that
SF162 is a V3-sensitive virus (12), and our results are consis-
tent with this observation. In contrast, there was no detectable
reduction of serum neutralizing activity against HXBc2 (Fig. 4,
middle panel) in the V3 peptide competition assay and only
sporadic reduction against MW965 (data not shown), perhaps
due to V3 mismatch. Additional attempts to map the neutral-
izing antibody response against HXBc2 were made using a
peptide that spans the linear epitope of the gp41-broadly neu-
tralizing antibody 2F5 to probe for antibodies directed against
the membrane-proximal external region of gp41, which con-
tains the 2F5 epitope. No inhibition of neutralization was ob-
served in any of the 2F5-related mapping experiments (data
not shown). It is possible that antibodies recognizing the CD4-
induced coreceptor binding site of gp120 are responsible for
the neutralizing activity observed against HXBc2, since such
antibodies were detected in the sera from these animals (16a).
As mentioned above, the overall neutralizing activities were
qualitatively quite similar in three groups, but the magnitude of
the responses was somewhat lower in group 2 than in group 1
even at time points when the gp120 ELISA binding titers were
comparable. This prompted us to ask if the proportion of
antibodies recognizing denatured gp120 was greater in group 2
than in the other groups. It is possible, for example, that im-
mature, misfolded Env released from dying infected cells con-
tributed to the elicitation of antibody responses in rSFV-
primed animals. To address this, we analyzed all post-5 sera in
an ELISA using denatured gp120 antigen for coating side-by-
side with the conventional ELISA using native gp120 for coat-
ing (Fig. 5A). As controls, we used two monoclonal antibodies:
F105, which recognizes a conformation-sensitive epitope in
gp120, and D7324, which recognizes a linear epitope in the
C-terminal region of gp120. F105 was used to confirm that the
gp120 used for coating was denatured, and D7324 confirmed
FIG. 4. V3 peptide competition mapping of neutralizing activity. V3
peptide mapping was performed on individual serum samples against
MN, HXBc2, and SF162. The V3 peptide sequence used to map neutral-
izing activity targeting the V3 loop was based on the YU2 sequence
(TRPNNNTRKSINIGPGRALYTTG) (filled circles). A scrambled V3
peptide (IGPGRATRPNNNFYTTGTRKSIH) was used as a negative
control (open circles). The results from these experiments are shown as
50% are considered a reliable signal. Values in excess of a 50% reduction
of ID50neutralization are indicated by the dotted line.
546MO ¨RNER ET AL.J. VIROL.
that similar levels of protein were used in the two assays
(see Fig S3 in the supplemental material). Using this assay,
there was no marked difference between the sera in their
relative ability to recognize misfolded gp120 compared to the
We next asked why the regimen used in group 3 (stable core
priming, gp140-F trimer boosting) did not yield sera possessing
a greater increase in the breadth of neutralization. The ratio-
nale behind this regimen was that common epitopes between
the two proteins would allow elicitation (by the priming) and
selective expansion (by the boosting) of the B-cell response
recognizing conserved surfaces, which might translate into in-
creased neutralization breadth. Since this was not observed, we
performed an ELISA using either the stable core protein or
gp140-F trimers for coating, and we analyzed the sera obtained
from the animals in group 1 and group 3 after the second and
fourth immunizations. The results are presented as absorbance
against log reciprocal serum dilution for group means (Fig.
5B). When the stable core was used for coating, there was only
a modest boosting effect by the gp140-F trimers in group 3,
while in contrast, when the trimers were used for coating, there
was a substantial boost effect by the trimers in group 3. These
data suggest that perhaps the cross-reactivity between the sta-
ble core protein and gp140-F is limited and that the increase in
titers after gp140-F trimer immunizations likely represents new
reactivities against trimer-specific determinants rather than
boosting of memory B cells initially stimulated by the stable
core prime. Nevertheless, trimers were able to modestly boost
the stable core-directed antibody responses, suggesting that
the strategy of immunofocusing may be beneficial with an
improved immunogen design (37).
Env-specific T-cell responses in immunized macaques. An
additional goal of this study was to analyze Env-specific T-cell
responses and particularly to determine whether priming with
the stable core protein conferred an advantage in focusing the
T-cell responses on more conserved regions of Env. Env-spe-
cific T-cell responses were measured using an IFN-? ELISPOT
assay with cells isolated before immunization or after the sec-
ond or fourth immunization. Total PBMC were stimulated
with two pools of 15-mer peptides spanning the gp140-F pro-
tein sequence. The “gp140-F core” peptide pool covered the
conserved regions of gp120, corresponding to the stable core
immunogen, while the “gp140-F noncore” peptide pool cov-
ered the remaining parts of gp140. Results are shown as total
spot-forming cells per 1 million PBMC (Fig. 6A). High Env-
specific IFN-? responses were obtained in all three groups, and
a clear increase in the magnitude of the responses was mea-
sured between the second and fourth immunizations. These
data demonstrate the ability of gp140-F trimers administered
in the AS01B adjuvant system to boost T-cell responses elicited
either by prior immunizations with protein or by rSFV-gp140-F
The most striking outcome from the T-cell analysis was the
clear difference in the response to the different peptide pools
observed between the groups (Fig. 6A). The animals in group
1 and group 2 had a dominant IFN-? response to the noncore
peptide pool and a relatively weak response to the core peptide
pool. In contrast, IFN-? responses to the core peptide pool
dominated in group 3 even after two boosts with gp140-F
trimers with no or very low responses against the noncore
pools. These data demonstrate very clearly that heterologous
prime-boost regimens can be used to focus the T-cell response
on conserved epitopes of HIV-1 Env. When the frequencies of
core-specific responses of total Env-specific T-cell responses
were analyzed, the animals in group 1 and group 2 showed
similar patterns despite the two different platforms by which
the trimers were administered (Fig. 6B). In contrast, the ani-
mals in group 3 were strikingly different, with 80 to 100% of the
IFN-? T-cell responses directed against the core (Fig. 6B).
Similar results were obtained when the HXBc2 core peptide
pool was used for stimulation (data not shown), indicating that
FIG. 5. ELISA using different Env proteins for coating. (A) Serum
samples from 2 weeks after the fifth immunization were tested in an
ELISA using either native or denatured gp120 for coating. The native
versus denatured OD50titer ratio for each individual animal is shown.
(B) Binding antibodies against the stable core protein (upper panel) or
gp140-F (lower panel) used for coating in the ELISA plates were
measured in serum samples from individual animals 2 weeks after the
second and fourth immunizations and plotted as group means. “T”
refers to gp140-F trimer protein immunization, “S” refers to SFV-
gp140-F immunization, and “C” refers to stable core protein immuni-
VOL. 83, 2009 IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES547
the measured differences in the distribution of the core-specific
responses were not biased by the use of YU2 peptides.
To determine the relative contributions of CD4?versus
CD8?T cells for the overall Env-specific IFN-? T-cell re-
sponses, the CD8?T-cell fraction was depleted using magnetic
bead separation and the IFN-? ELISPOT assay was repeated
using the total PBMC fraction and the fraction depleted of
CD8?T cells (Fig. 6C). This resulted in an unaltered or in-
creased IFN-? response for all animals, suggesting that CD4?
T cells were responsible for the IFN-? response. The increased
response can be explained by a greater relative number of
CD4?T cells in the PBMC population after CD8?T-cell
depletion, indicating that the AS01B adjuvant system potently
induced Env-specific CD4?T-cell responses.
In this study, we characterized neutralizing antibody and
T-cell responses elicited by soluble YU2-based gp140-F tri-
mers administered in the AS01B adjuvant system (GSK) in
nonhuman primates. We also examined the consequences of
priming with a heterologous immunogen, either an alphavirus
replicon vector expressing matched gp140-F trimers or a mono-
meric gp120 core protein stabilized in the CD4-bound confor-
mation, the latter administered in the AS01B adjuvant system.
The stable core protein was selected in an attempt to focus the
immune response on conserved determinants of Env that are
common between the stable core and the gp140-F proteins.
High titers of antibody were elicited with primates immunized
with soluble gp140-F trimers in adjuvant, with OD50titers of
approximately 104, which corresponds to endpoint titers of
about 106, at the peak of the response. Protein trimers in the
AS01B adjuvant system also efficiently boosted antibody re-
sponses primed by rSFV-expressed gp140-F trimers, demon-
strating that this adjuvant also is highly efficacious following a
viral vector prime. The stable core protein appeared less im-
munogenic than the trimers, as measured by lower antibody
responses after the first and second immunizations. We ini-
tially hypothesized that this might be explained by fewer CD4
T-cell helper epitopes in the stable cores than in the trimer
proteins, but our subsequent T-cell analysis showed that the
T-cell response against the stable core protein was in fact
higher than that observed against the trimer, as discussed
Attempts to focus the immune response on shared neutral-
izing determinants between the stable core and the gp140-F
trimers did not markedly improve the neutralizing antibody
response. One possible explanation for this is that broadly
neutralizing determinants, such as the CD4 binding site, are
not similar enough between the two proteins. For example,
both proteins bind CD4, but the stable cores are, by design,
deficient in their ability to bind many of the nonneutralizing
CD4-binding site antibodies. Also, this version of the stable
core was reported to display a slightly decreased affinity for the
broadly neutralizing CD4 binding site antibody, b12, as well as
a somewhat increased affinity for CD4 itself (56). These
changes in ligand affinities for the CD4-binding site might
FIG. 6. Vaccine-induced T-cell responses. HIV-1 Env-directed T-cell responses were measured by IFN-? ELISPOT after restimulation of
PBMC with overlapping peptides. Two different peptide pools based on the YU2 gp140 sequence were used: “gp140-F core” covered the conserved
regions of gp120, corresponding to the stable core immunogen, and “gp140-F non-core” covered the remaining parts of YU2 gp140. (A) gp140-F
core (light purple) and noncore (dark purple) responses before immunization (Pre) and 2 weeks after the second (2?T) and fourth (4?T)
immunizations are shown. “T” refers to gp140-F trimer protein immunization, “S” refers to SFV-gp140-F immunization, and “C” refers to stable
core protein immunization. (B) The percent core-specific response of the total gp140-F response after the second and fourth immunizations is
shown. (C) PBMC collected 2 weeks after immunization 4 were depleted of CD8?T cells by magnetic cell sorting. Total PBMC (PBMC) and CD8?
T-cell-depleted PBMC (?CD8) were restimulated with the gp140-F core and gp140-F noncore peptide pools. Shown are the cumulative gp140-F
core and noncore responses. The viability in both total PBMC and CD8?T-cell-depleted PBMC was ?93%. The CD8?T-cell-depleted PBMC
contained ?0.2% CD8?T cells, as measured by flow cytometry.
548MO ¨RNER ET AL. J. VIROL.
indicate that at least a portion of this surface is different be-
tween the core and the trimer. Poor cross-reactivity between
the stable cores and gp140-F was also observed in the ELISA
analysis shown in Fig. 5B, which demonstrates that gp140-F
immunizations poorly boosted the stable core-specific antibody
responses. In contrast, responses against the trimer were mark-
edly increased by gp140-F immunizations (Fig. 5B), suggesting
stimulation of new antibody reactivities, such as those directed
against the immunogenic variable regions of Env, rather than
boosting of responses previously elicited by the stable cores.
Although not studied here, longer intervals that lead to a
waning of the serum antibody levels between each immuniza-
tion may also favorably affect the distribution of the memory
B-cell compartment, with an impact on the expansion of B-cell
clones that recognize common determinants between the pro-
Binding antibody titers against gp120 after a single rSFV-
gp140-F immunization were surprisingly high, reaching OD50
titers of between 102and 103, and this response was enhanced
by subsequent inoculations of gp140-F trimers in AS10B.
Overall, responses in the three groups were qualitatively sim-
ilar after the gp140-F trimer boosts. Significant neutralization
titers were detected against HXBc2, MN, SF162 (all clade B),
and the clade C virus MW965. Our results with the gp140-F
trimers are consistent with the results obtained in other recent
studies with nonhuman primates using the TZM-bl-cell single-
cycle infectivity assay (42). ID50titers against SF162 were
around 100 to 1,000, which is the same range as the responses
associated with protection against vaginal SHIV-SF162P4 chal-
lenge (2). We found that a considerable fraction of the neu-
tralizing activity against SF162 and MN was mediated by an-
tibodies against the V3 region of Env, as determined by
peptide competition studies, consistent with cross-reactivity
between the YU2 and the V3 region of these viruses. Since the
majority of V3-directed antibodies tend to be strain specific
rather than broadly neutralizing and most primary isolates are
selected to occlude their V3 loops, a dominant antibody re-
sponse against the V3 region is not desired for a broadly
protective HIV-1 vaccine. Modifications designed to dampen
the V3-directed response elicited by the gp140-F trimers are
therefore warranted, such as the hyperglycosylation strategies
described by Selvarajah et al. (43, 44).
We observed a striking difference in the specificities of the
Env-specific T-cell responses when the three groups were com-
pared side-by-side using pools of peptides spanning core versus
noncore regions of the gp140-F trimers. While the dominant
response in groups 1 and 2 was directed against epitopes in the
noncore peptide pool, the response in group 3, even after the
gp140-F boost, was almost exclusively (around 90%) directed
against epitopes contained in the core peptide pool. These data
highlight the potential of priming with Env core proteins to
focus the cellular response on conserved regions of Env, pre-
sumably by increasing the frequencies of primed T cells against
the conserved core determinants. Strategies to focus on the
immune response in such a manner may add value to current
attempts to induce protective immune responses to HIV-1.
Finally, we also asked if there was a difference between the
groups in terms of how much of the response was mediated by
CD4?T cells compared to CD8?T cells. Detectable Env-
directed IFN-? T-cell responses appeared to be mediated by
CD4?T-cell responses in all three groups.
In conclusion, the inability of the core prime/trimer boost
approach to broaden the neutralizing antibody responses may
suggest that the stable core protein shares too few common
immunogenic B-cell epitopes with gp140-F or that one or both
of these immunogens are insufficient mimics of the functional
viral spike complex. However, a marked shift in the specificity
of the T-cell response was observed with this regimen. The
utility of Env as a T-cell immunogen was recently called into
question (36). Perhaps creative and rational attempts to focus
both the B- and T-cell responses on conserved determinants of
Env, such as those presented here, are worthy of consideration
in the design of vaccine regimens capable of stimulating pro-
tective immune responses against HIV-1.
We thank Mats Spångberg, Helene Fredlund, and the personnel at
Astrid Fagraeus laboratory at the Swedish Institute for Infectious
Disease Control for expert assistance and Eva Hansson Pihlainen,
Ulrika Edba ¨ck, and Irene Silhammar for skilled technical assistance.
We also thank Chih-chin Huang for contributions to the structure-
based additions to V3 in the stable core context and Brenda Hartman,
Mike Cichanowski, and Jonathan Stuckey for help with Fig. 1.
This study was supported by a grant from the Swedish International
Development Agency (Sida)/Department of Research Cooperation
(SAREC) to G.K.H. and R.T. and by the National Institute of Allergy
and Infectious Diseases, National Institutes of Health intramural re-
search program, for P.D.K., J.R.M., and R.T.W. Funding was also
received from the Bill and Melinda Gates Foundation (P.D.K., J.R.M.,
and R.T.W.), the International AIDS Vaccine Initiative (P.D.K.,
R.T.W., and G.K.H.), and the Swedish Research Council (G.K.H.).
1. Barnett, S. W., S. Lu, I. Srivastava, S. Cherpelis, A. Gettie, J. Blanchard, S.
Wang, I. Mboudjeka, L. Leung, Y. Lian, A. Fong, C. Buckner, A. Ly, S. Hilt,
J. Ulmer, C. T. Wild, J. R. Mascola, and L. Stamatatos. 2001. The ability of
an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope an-
tigen to elicit neutralizing antibodies against primary HIV-1 isolates is im-
proved following partial deletion of the second hypervariable region. J. Vi-
2. Barnett, S. W., I. K. Srivastava, E. Kan, F. Zhou, A. Goodsell, A. D. Cristillo,
M. G. Ferrai, D. E. Weiss, N. L. Letvin, D. Montefiori, R. Pal, and M. Vajdy.
2008. Protection of macaques against vaginal SHIV challenge by systemic or
mucosal and systemic vaccinations with HIV-envelope. AIDS 22:339–348.
3. Berglund, P., M. N. Fleeton, C. Smerdou, and P. Liljestrom. 1999. Immu-
nization with recombinant Semliki Forest virus induces protection against
influenza challenge in mice. Vaccine 17:497–507.
4. Berglund, P., M. Quesada-Rolander, P. Putkonen, G. Biberfeld, R. Thor-
stensson, and P. Liljestrom. 1997. Outcome of immunization of cynomolgus
monkeys with recombinant Semliki Forest virus encoding human immuno-
deficiency virus type 1 envelope protein and challenge with a high dose of
SHIV-4 virus. AIDS Res. Hum. Retrovir. 13:1487–1495.
5. Binley, J. M., R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F.
Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore. 2000.
A recombinant human immunodeficiency virus type 1 envelope glycoprotein
complex stabilized by an intermolecular disulfide bond between the gp120
and gp41 subunits is an antigenic mimic of the trimeric virion-associated
structure. J. Virol. 74:627–643.
6. Bower, J. F., X. Yang, J. Sodroski, and T. M. Ross. 2004. Elicitation of
neutralizing antibodies with DNA vaccines expressing soluble stabilized hu-
man immunodeficiency virus type 1 envelope glycoprotein trimers conju-
gated to C3d. J. Virol. 78:4710–4719.
7. Burton, D. R., R. C. Desrosiers, R. W. Doms, W. C. Koff, P. D. Kwong, J. P.
Moore, G. J. Nabel, J. Sodroski, I. A. Wilson, and R. T. Wyatt. 2004. HIV
vaccine design and the neutralizing antibody problem. Nat. Immunol. 5:233–
8. Caley, I. J., M. R. Betts, N. L. Davis, R. Swanstrom, J. A. Frelinger, and R. E.
Johnston. 1999. Venezuelan equine encephalitis virus vectors expressing
HIV-1 proteins: vector design strategies for improved vaccine efficacy. Vac-
9. Catanzaro, A. T., M. Roederer, R. A. Koup, R. T. Bailer, M. E. Enama, M. C.
Nason, J. E. Martin, S. Rucker, C. A. Andrews, P. L. Gomez, J. R. Mascola,
VOL. 83, 2009 IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES 549
G. J. Nabel, and B. S. Graham. 2007. Phase I clinical evaluation of a
six-plasmid multiclade HIV-1 DNA candidate vaccine. Vaccine 25:4085–
10. Chen, Z., Y. Huang, X. Zhao, L. Ba, W. Zhang, and D. D. Ho. 2008. Design,
construction, and characterization of a multigenic modified vaccinia Ankara
candidate vaccine against human immunodeficiency virus type 1 subtype
C/B?. J. Acquir. Immune Defic Syndr. 47:412–421.
11. Davis, N. L., A. West, E. Reap, G. MacDonald, M. Collier, S. Dryga, M.
Maughan, M. Connell, C. Walker, K. McGrath, C. Cecil, L. H. Ping, J.
Frelinger, R. Olmsted, P. Keith, R. Swanstrom, C. Williamson, P. Johnson,
D. Montefiori, and R. E. Johnston. 2002. Alphavirus replicon particles as
candidate HIV vaccines. IUBMB Life 53:209–211.
12. Derby, N. R., Z. Kraft, E. Kan, E. T. Crooks, S. W. Barnett, I. K. Srivastava,
J. M. Binley, and L. Stamatatos. 2006. Antibody responses elicited in
macaques immunized with human immunodeficiency virus type 1 (HIV-1)
SF162-derived gp140 envelope immunogens: comparison with those elicited
during homologous simian/human immunodeficiency virus SHIVSF162P4
and heterologous HIV-1 infection. J. Virol. 80:8745–8762.
13. Dey, B., M. Pancera, K. Svehla, Y. Shu, S. H. Xiang, J. Vainshtein, Y. Li, J.
Sodroski, P. D. Kwong, J. R. Mascola, and R. Wyatt. 2007. Characterization
of human immunodeficiency virus type 1 monomeric and trimeric gp120
glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and
immunogenicity. J. Virol. 81:5579–5593.
14. Fleeton, M. N., M. Chen, P. Berglund, G. Rhodes, S. E. Parker, M. Murphy,
G. J. Atkins, and P. Liljestrom. 2001. Self-replicative RNA vaccines elicit
protection against influenza A virus, respiratory syncytial virus, and a tick-
borne encephalitis virus. J. Infect. Dis. 183:1395–1398.
15. Forsell, M. N., Y. Li, M. Sundback, K. Svehla, P. Liljestrom, J. R. Mascola,
R. Wyatt, and G. B. Hedestam. 2005. Biochemical and immunogenic char-
acterization of soluble human immunodeficiency virus type 1 envelope gly-
coprotein trimers expressed by Semliki Forest virus. J. Virol. 79:10902–
16. Forsell, M. N., G. M. McInerney, P. Dosenovic, A. S. Hidmark, C. Eriksson,
P. Liljestrom, C. Grundner, and G. B. Karlsson Hedestam. 2007. Increased
human immunodeficiency virus type 1 Env expression and antibody induc-
tion using an enhanced alphavirus vector. J. Gen. Virol. 88:2774–2779.
16a.Forsell, M. N. E., B. Dey, A. Mo ¨rner, K. Svehla, S. O’dell, C.-M. Ho ¨gerkorp,
G. Voss, R. Thorstensson, G. M. Shaw, J. R. Mascola, G. B. Karlsson
Hedestam, and R. T. Wyatt. 2008. B cell recognition of the conserved HIV-I
co-receptor binding site is altered by endogenous primate CD4. PLoS
Pathog. 4:c1000171. doi:10.1371/journal.ppat.1000171.
17. Gorny, M. K., A. J. Conley, S. Karwowska, A. Buchbinder, J. Y. Xu, E. A.
Emini, S. Koenig, and S. Zolla-Pazner. 1992. Neutralization of diverse hu-
man immunodeficiency virus type 1 variants by an anti-V3 human mono-
clonal antibody. J. Virol. 66:7538–7542.
18. Harari, A., P. A. Bart, W. Stohr, G. Tapia, M. Garcia, E. Medjitna-Rais, S.
Burnet, C. Cellerai, O. Erlwein, T. Barber, C. Moog, P. Liljestrom, R.
Wagner, H. Wolf, J. P. Kraehenbuhl, M. Esteban, J. Heeney, M. J. Frachette,
J. Tartaglia, S. McCormack, A. Babiker, J. Weber, and G. Pantaleo. 2008.
An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces
reliable, polyfunctional, and long-lasting T cell responses. J. Exp. Med.
19. Hidmark, A. S., G. M. McInerney, E. K. Nordstrom, I. Douagi, K. M.
Werner, P. Liljestrom, and G. B. Hedestam. 2005. Early alpha/beta inter-
feron production by myeloid dendritic cells in response to UV-inactivated
virus requires viral entry and interferon regulatory factor 3 but not MyD88.
J. Virol. 79:10376–10385.
20. Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L.
Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and
P. D. Kwong. 2005. Structure of a V3-containing HIV-1 gp120 core. Science
21. Huckriede, A., L. Bungener, M. Holtrop, J. de Vries, B. L. Waarts, T.
Daemen, and J. Wilschut. 2004. Induction of cytotoxic T lymphocyte activity
by immunization with recombinant Semliki Forest virus: indications for
cross-priming. Vaccine 22:1104–1113.
22. Karlsson, G. B., and P. Liljestrom. 2003. Live viral vectors: Semliki Forest
virus. Methods Mol. Med. 87:69–82.
23. Karlsson Hedestam, G. B., R. A. Fouchier, S. Phogat, D. R. Burton, J.
Sodroski, and R. T. Wyatt. 2008. The challenges of eliciting neutralizing
antibodies to HIV-1 and to influenza virus. Nat. Rev. Microbiol. 6:143–155.
24. Kim, M., Z. S. Qiao, D. C. Montefiori, B. F. Haynes, E. L. Reinherz, and
H. X. Liao. 2005. Comparison of HIV type 1 ADA gp120 monomers versus
gp140 trimers as immunogens for the induction of neutralizing antibodies.
AIDS Res. Hum. Retrovir. 21:58–67.
25. Letvin, N. L., Y. Huang, B. K. Chakrabarti, L. Xu, M. S. Seaman, K.
Beaudry, B. Korioth-Schmitz, F. Yu, D. Rohne, K. L. Martin, A. Miura, W. P.
Kong, Z. Y. Yang, R. S. Gelman, O. G. Golubeva, D. C. Montefiori, J. R.
Mascola, and G. J. Nabel. 2004. Heterologous envelope immunogens con-
tribute to AIDS vaccine protection in rhesus monkeys. J. Virol. 78:7490–
26. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos,
G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, M. Bilska, D. L. Kothe, J. F.
Salazar-Gonzalez, X. Wei, J. M. Decker, B. H. Hahn, and D. C. Montefiori.
2005. Human immunodeficiency virus type 1 env clones from acute and early
subtype B infections for standardized assessments of vaccine-elicited neu-
tralizing antibodies. J. Virol. 79:10108–10125.
27. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X.
Wu, G. M. Shaw, M. Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad
HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med.
28. Li, Y., K. Svehla, N. L. Mathy, G. Voss, J. R. Mascola, and R. Wyatt. 2006.
Characterization of antibody responses elicited by human immunodeficiency
virus type 1 primary isolate trimeric and monomeric envelope glycoproteins
in selected adjuvants. J. Virol. 80:1414–1426.
29. Morris-Downes, M. M., K. V. Phenix, J. Smyth, B. J. Sheahan, S. Lileqvist,
D. A. Mooney, P. Liljestrom, D. Todd, and G. J. Atkins. 2001. Semliki Forest
virus-based vaccines: persistence, distribution and pathological analysis in
two animal systems. Vaccine 19:1978–1988.
30. Morris-Downes, M. M., B. J. Sheahan, M. N. Fleeton, P. Liljestrom, H. W.
Reid, and G. J. Atkins. 2001. A recombinant Semliki Forest virus particle
vaccine encoding the prME and NS1 proteins of louping ill virus is effective
in a sheep challenge model. Vaccine 19:3877–3884.
31. Mossman, S. P., F. Bex, P. Berglund, J. Arthos, S. P. O’Neil, D. Riley, D. H.
Maul, C. Bruck, P. Momin, A. Burny, P. N. Fultz, J. I. Mullins, P. Liljestrom,
and E. A. Hoover. 1996. Protection against lethal simian immunodeficiency
virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160
vaccine and by a gp120 subunit vaccine. J. Virol. 70:1953–1960.
32. Nordstrom, E. K., M. N. Forsell, C. Barnfield, E. Bonin, T. Hanke, M.
Sundstrom, G. B. Karlsson, and P. Liljestrom. 2005. Enhanced immunoge-
nicity using an alphavirus replicon DNA vaccine against human immunode-
ficiency virus type 1. J. Gen. Virol. 86:349–354.
33. Pantophlet, R., and D. R. Burton. 2006. GP120: target for neutralizing
HIV-1 antibodies. Annu. Rev. Immunol. 24:739–769.
34. Patterson, L. J., N. Malkevitch, D. Venzon, J. Pinczewski, V. R. Gomez-
Roman, L. Wang, V. S. Kalyanaraman, P. D. Markham, F. A. Robey, and M.
Robert-Guroff. 2004. Protection against mucosal simian immunodeficiency
virus SIVmac251challenge by using replicating adenovirus-SIV multigene
vaccine priming and subunit boosting. J. Virol. 78:2212–2221.
35. Perri, S., C. E. Greer, K. Thudium, B. Doe, H. Legg, H. Liu, R. E. Romero,
Z. Tang, Q. Bin, T. W. Dubensky, Jr., M. Vajdy, G. R. Otten, and J. M. Polo.
2003. An alphavirus replicon particle chimera derived from Venezuelan
equine encephalitis and Sindbis viruses is a potent gene-based vaccine de-
livery vector. J. Virol. 77:10394–10403.
36. Peut, V., and S. J. Kent. 2007. Utility of human immunodeficiency virus type
1 envelope as a T-cell immunogen. J. Virol. 81:13125–13134.
37. Phogat, S., and R. Wyatt. 2007. Rational modifications of HIV-1 envelope
glycoproteins for immunogen design. Curr. Pharm. Des. 13:213–227.
38. Priddy, F. H., D. Brown, J. Kublin, K. Monahan, D. P. Wright, J. Lalezari,
S. Santiago, M. Marmor, M. Lally, R. M. Novak, S. J. Brown, P. Kulkarni,
S. A. Dubey, L. S. Kierstead, D. R. Casimiro, R. Mogg, M. J. DiNubile, J. W.
Shiver, R. Y. Leavitt, M. N. Robertson, D. V. Mehrotra, and E. Quirk. 2008.
Safety and immunogenicity of a replication-incompetent adenovirus type 5
HIV-1 clade B gag/pol/nef vaccine in healthy adults. Clin. Infect. Dis. 46:
39. Robinson, H. L., S. Sharma, J. Zhao, S. Kannanganat, L. Lai, L.
Chennareddi, T. Yu, D. C. Montefiori, R. R. Amara, L. S. Wyatt, and B.
Moss. 2007. Immunogenicity in macaques of the clinical product for a clade
B DNA/MVA HIV vaccine: elicitation of IFN-gamma, IL-2, and TNF-alpha
coproducing CD4 and CD8 T cells. AIDS Res. Hum. Retrovir. 23:1555–
40. Sanders, R. W., M. Vesanen, N. Schuelke, A. Master, L. Schiffner, R. Kaly-
anaraman, M. Paluch, B. Berkhout, P. J. Maddon, W. C. Olson, M. Lu, and
J. P. Moore. 2002. Stabilization of the soluble, cleaved, trimeric form of the
envelope glycoprotein complex of human immunodeficiency virus type 1.
J. Virol. 76:8875–8889.
41. Schlesinger, S., and T. W. Dubensky. 1999. Alphavirus vectors for gene
expression and vaccines. Curr. Opin. Biotechnol. 10:434–439.
42. Seaman, M. S., D. F. Leblanc, L. E. Grandpre, M. T. Bartman, D. C.
Montefiori, N. L. Letvin, and J. R. Mascola. 2007. Standardized assessment
of NAb responses elicited in rhesus monkeys immunized with single- or
multi-clade HIV-1 envelope immunogens. Virology 367:175–186.
43. Selvarajah, S., B. Puffer, R. Pantophlet, M. Law, R. W. Doms, and D. R.
Burton. 2005. Comparing antigenicity and immunogenicity of engineered
gp120. J. Virol. 79:12148–12163.
44. Selvarajah, S., B. A. Puffer, F. H. Lee, P. Zhu, Y. Li, R. Wyatt, K. H. Roux,
R. W. Doms, and D. R. Burton. 2008. Focused dampening of antibody
response to the immunodominant variable loops by engineered soluble
gp140. AIDS Res. Hum. Retrovir. 24:301–314.
45. Shu, Y., S. Winfrey, Z. Y. Yang, L. Xu, S. S. Rao, I. Srivastava, S. W. Barnett,
G. J. Nabel, and J. R. Mascola. 2007. Efficient protein boosting after plasmid
DNA or recombinant adenovirus immunization with HIV-1 vaccine con-
structs. Vaccine 25:1398–1408.
46. Srivastava, I. K., L. Stamatatos, E. Kan, M. Vajdy, Y. Lian, S. Hilt, L.
Martin, C. Vita, P. Zhu, K. H. Roux, L. Vojtech, C. D. Montefiori, J.
550 MO ¨RNER ET AL.J. VIROL.
Donnelly, J. B. Ulmer, and S. W. Barnett. 2003. Purification, characteriza- Download full-text
tion, and immunogenicity of a soluble trimeric envelope protein containing
a partial deletion of the V2 loop derived from SF162, an R5-tropic human
immunodeficiency virus type 1 isolate. J. Virol. 77:11244–11259.
47. Sundback, M., I. Douagi, C. Dayaraj, M. N. Forsell, E. K. Nordstrom, G. M.
McInerney, K. Spangberg, L. Tjader, E. Bonin, M. Sundstrom, P.
Liljestrom, and G. B. Karlsson Hedestam. 2005. Efficient expansion of
HIV-1-specific T cell responses by homologous immunization with recom-
binant Semliki Forest virus particles. Virology 341:190–202.
48. Thongcharoen, P., V. Suriyanon, R. M. Paris, C. Khamboonruang, M. S. de
Souza, S. Ratto-Kim, C. Karnasuta, V. R. Polonis, L. Baglyos, R. E. Habib,
S. Gurunathan, S. Barnett, A. E. Brown, D. L. Birx, J. G. McNeil, and J. H.
Kim. 2007. A phase 1/2 comparative vaccine trial of the safety and immu-
nogenicity of a CRF01_AE (subtype E) candidate vaccine: ALVAC-HIV
(vCP1521) prime with oligomeric gp160 (92TH023/LAI-DID) or bivalent
gp120 (CM235/SF2) boost. J. Acquir. Immune Defic. Syndr. 46:48–55.
49. Wang, S., J. S. Kennedy, K. West, D. C. Montefiori, S. Coley, J. Lawrence, S.
Shen, S. Green, A. L. Rothman, F. A. Ennis, J. Arthos, R. Pal, P. Markham,
and S. Lu. 2008. Cross-subtype antibody and cellular immune responses
induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy
human volunteers. Vaccine 26:1098–1110.
50. Xiang, S. H., P. D. Kwong, R. Gupta, C. D. Rizzuto, D. J. Casper, R. Wyatt,
L. Wang, W. A. Hendrickson, M. L. Doyle, and J. Sodroski. 2002. Mutagenic
stabilization and/or disruption of a CD4-bound state reveals distinct confor-
mations of the human immunodeficiency virus type 1 gp120 envelope glyco-
protein. J. Virol. 76:9888–9899.
51. Xu, R., I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M.
Polo, S. W. Barnett, and L. Stamatatos. 2006. Characterization of immune
responses elicited in macaques immunized sequentially with chimeric VEE/
SIN alphavirus replicon particles expressing SIVGag and/or HIVEnv and
with recombinant HIVgp140Env protein. AIDS Res. Hum. Retrovir. 22:
52. Xu, R., I. K. Srivastava, L. Kuller, I. Zarkikh, Z. Kraft, Z. Fagrouch, N. L.
Letvin, J. L. Heeney, S. W. Barnett, and L. Stamatatos. 2006. Immunization
with HIV-1 SF162-derived envelope gp140 proteins does not protect
SHIV89.6P infection. Virology 349:276–289.
53. Yang, X., M. Farzan, R. Wyatt, and J. Sodroski. 2000. Characterization of
stable, soluble trimers containing complete ectodomains of human immu-
nodeficiency virus type 1 envelope glycoproteins. J. Virol. 74:5716–5725.
54. Yang, X., J. Lee, E. M. Mahony, P. D. Kwong, R. Wyatt, and J. Sodroski.
2002. Highly stable trimers formed by human immunodeficiency virus type 1
envelope glycoproteins fused with the trimeric motif of T4 bacteriophage
fibritin. J. Virol. 76:4634–4642.
55. Yang, X., R. Wyatt, and J. Sodroski. 2001. Improved elicitation of neutral-
izing antibodies against primary human immunodeficiency viruses by soluble
stabilized envelope glycoprotein trimers. J. Virol. 75:1165–1171.
56. Zhou, T., L. Xu, B. Dey, A. J. Hessell, D. Van Ryk, S. H. Xiang, X. Yang,
M. Y. Zhang, M. B. Zwick, J. Arthos, D. R. Burton, D. S. Dimitrov, J.
Sodroski, R. Wyatt, G. J. Nabel, and P. D. Kwong. 2007. Structural definition
of a conserved neutralization epitope on HIV-1 gp120. Nature 445:732–737.
VOL. 83, 2009IMMUNOGENICITY OF HIV-1 Env VARIANTS IN MACAQUES551