JOURNAL OF VIROLOGY, Feb. 2006, p. 1414–1426
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 3
Characterization of Antibody Responses Elicited by Human
Immunodeficiency Virus Type 1 Primary Isolate Trimeric and
Monomeric Envelope Glycoproteins in Selected Adjuvants†
Y. Li,1K. Svehla,1N. L. Mathy,2G. Voss,2J. R. Mascola,1and R. Wyatt1*
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland,1and GlaxoSmithKline Biologicals, Rixensart, Belgium2
Received 1 August 2005/Accepted 26 August 2005
We previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1
(HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1
isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to
extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing
response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140
immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than
those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A,
and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens
in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not
mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some
heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was
inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140
trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response,
sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as deter-
mined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutral-
izing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis
of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by
the trimeric immunogens.
The human immunodeficiency virus type 1 (HIV-1) exterior
envelope glycoprotein, gp120, mediates entry by binding to the
viral primary receptor CD4 (8, 29, 38) and the coreceptors
CCR5 (1, 7, 9, 11, 12, 18) or CXCR4 (50, 51, 54). The trans-
membrane glycoprotein, gp41, contains the oligomerization
domain (5, 58) and mediates virus-to-cell membrane fusion.
These glycoproteins are derived from gp160 precursor proteins
that, following glycosylation, folding, and trimerization in the
endoplasmic reticulum-Golgi, are cleaved into the nonco-
valently associated gp120-gp41 heterodimeric, trimeric spikes
(2, 14, 15, 35, 45, 48, 57). Due to their exposed location on the
surface of the virus (or infected cells), the gp120 and gp41
proteins are the sole viral targets for neutralizing antibodies.
Since effective neutralizing antibodies are likely to be a critical
component of a successful HIV vaccine, a great deal of effort
has focused on how to more efficiently elicit antibodies of
breadth and potency capable of neutralizing a broad array of
primary isolates. The first clinical trial utilizing monomeric
gp120 as an immunogen failed to demonstrate any level of
protection (19); hence, the focus has shifted to design of mol-
ecules that more closely resemble the trimeric spike found on
the virus (3, 4, 13, 16, 20, 27, 52, 59–61).
We previously reported that gp140 (?/GCN4) trimeric im-
munogens could elicit improved, although limited, breadth of
neutralization against HIV-1 isolates compared to monomeric
gp120 immunogens (22, 61). In this study, we sought to confirm
and extend these observations in another animal model and to
examine if adjuvant could further enhance the neutralizing
response. Thus, we compared YU2-based gp120 and gp140
immunogens emulsified in the commercially available Ribi ad-
juvant or in one of several newer adjuvants that have under-
gone extensive optimization with more modern technologies to
improve their efficacy.
Adjuvants function in at least two distinct ways. In a rela-
tively nonspecific manner, adjuvants increase the in vivo half-
life of the immunogen by a “depot effect” that increases the
persistence of the immunogen at the site of inoculation. Many
oil-in-water or water-in-oil adjuvants accomplish depot, or
deposition, through the formulation of an immunogen-con-
taining emulsion that slowly releases the protein immunogen
to interact with the host immune system. Adsorption of the
protein to alum precipitates provides another means to accom-
plish protein deposition, and currently alum still remains the
most widely used adjuvant for clinical applications.
Besides the “depot effect,” many adjuvants contain other
components that activate innate inflammatory and adaptive
responses, including humoral responses, by targeting known or
not-yet-defined “danger signal”-sensing receptors to improve
immunogenicity. For example, monophosphoryl lipid A
(MPL), the active component of lipopolysaccharide that inter-
* Corresponding author. Mailing address: Vaccine Research Center,
National Institutes of Health, 40 Convent Drive, Rm 4512, Bethesda,
MD 20892. Phone: (301) 594-8690. Fax: (301) 480-0274. E-mail:
† Supplemental material for this article may be found at http://jvi
acts with Toll-like receptor 4 (17, 39), is a component of Ribi
adjuvant and two of the other adjuvants tested here. In this
study, we analyzed antibody responses to the trimeric immu-
nogens compared to monomeric gp120. We also compared
Ribi adjuvant to three adjuvants developed by GlaxoSmithKline
Biologicals (GSK, Rixensart, Belgium), called AS01B, AS02A,
and AS03, to assess if the GSK adjuvants could elicit enhanced
immune responses to the immunogens. These adjuvants have
undergone extensive optimization to increase both humoral
and cell-mediated immunity (32, 53). Ribi adjuvant contains
the Toll-like receptor 4 agonist in a metabolizable oil, as well
as natural and synthesized microbial components. The adju-
vants from GSK are well defined and have been used in clinical
trials. GSK AS01B is comprised of liposomes, QS21 and
MPL™, GSK AS02A is an oil-in-water adjuvant containing
QS21 and MPL™, and GSK AS03 is composed of oil in water.
For each of the adjuvants tested in this study, the YU2
gp140(?/GCN4) immunogens elicit higher-titer neutralizing
antibodies than monomeric YU2 gp120. Mapping of the anti-
body responses indicates that neither immunogen elicits ho-
mologous V3-directed neutralizing responses, although signif-
icant levels of V3 loop antibodies are produced. The elicited
V3 loop antibodies appeared to neutralize some heterologous
isolates but not all. The homologous neutralization elicited by
YU2 gp120 is completely inhibited by a single peptide located
in the C-terminal region of the YU2 V1 loop. However, this is
not true for the limited heterologous neutralization activity
elicited by YU2 gp120. The homologous neutralizing activity
generated by YU2 gp140(?/GCN4) trimers is much less sen-
sitive to V1 peptide inhibition. This report identifies a novel
focus of the homologous neutralizing activity elicited by mo-
nomeric gp120 and outlines a nascent strategy to dissect the
complex binding and neutralizing specificities elicited by Env-
based immunogens. Although we can better map the neutral-
izing specificities elicited by the monomeric immunogens, the
trimeric immunogens elicit qualitatively different and more
diverse specificities. Further mapping analysis of more con-
served regions of gp120 or gp41 will be required to determine
neutralizing specificity elicited by the trimeric immunogens.
METHODS AND MATERIALS
Expression and purification of monomeric and trimeric envelope glycopro-
teins. The envelope glycoproteins were expressed by transfecting the 293F cell
line that has been adapted to serum-free medium (Invitrogen, Carlsbad, CA). In
brief, the 293F cells were seeded in T225 flasks at a density of 1 ? 105cells/cm2
and transfected with the expression plasmid YU2gp120/pcDNA3.1(?) for mo-
nomeric gp120 or YU2gp140(?/GCN4)/pcDNA3.1(?) DNA for trimeric
gp140(?/GCN4) proteins. Each of the encoded protein sequences contained
in-frame sequences encoding a six-His-tag at the C terminus as described pre-
viously (22). The cell culture supernatants were collected, centrifuged at 3,500 ?
g to remove cell debris, exchanged into phosphate buffer (20 mM phosphate, 0.5
M NaCl) with 10 mM imidazole, pH 7.4 by dialysis, and applied to a 5-ml
His-trap nickel affinity column (Amersham, Piscataway, NJ). For monomeric
gp120, the nickel affinity column was washed with phosphate buffer containing 10
mM imidazole, pH 7.4, and phosphate buffer containing 40 mM imidazole, pH
7.4, sequentially, and the proteins were eluted by phosphate buffer containing
300 mM imidazole. For trimeric gp140(?/GCN4) proteins, to minimize copuri-
fication of monomeric forms of gp140 in the culture supernatant, we increased
the wash stringency prior to elution. The nickel affinity column was washed with
phosphate buffer containing 30 mM imidazole, pH 7.4, followed by phosphate
buffer containing 60 mM imidazole, pH 7.4, and the protein oligomers were
eluted with phosphate buffer containing 300 mM imidazole. The protein eluates
were dialyzed against phosphate-buffered saline (PBS), pH 7.4, and concentrated
with Amicon Ultra 30,000 MWCO centrifugal filter devices (Millipore, Bedford,
MA). The purified proteins were subjected to sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis, blue native gel, and gel filtration analysis, and the
purity was verified to approach 95% homogeneity. By gel filtration, the gp140(?/
GCN4) proteins purified by this nickel affinity procedure contained no detectable
monomer and approximately 70% trimer; the remaining proteins were higher-
order oligomers (not shown).
Immunization protocol. Hartlan guinea pigs (females, ?7 weeks of age) were
inoculated intramuscularly with 20 ?g of either monomeric YU2 gp120 or trim-
eric YU2 gp140(?/GCN4) proteins emulsified in either 0.5 ml of Ribi (Corixa,
Hamilton, MT) or each of the GSK adjuvants. The protein-adjuvant emulsion
was always prepared within 1 h of inoculation into the animals. Boosting inoc-
ulations occurred 6, 10, 14, and 21 weeks after the initial inoculation. To isolate
serum, the blood was incubated at room temperature (RT) for 2 h to clot and
centrifuged for 10 min at 2000 ? g at RT to separate the liquid phase from the
clotted components. The serum was collected and incubated at 55°C for 1 h to
heat-inactivate complement and stored at ?20°C until subjected to analysis.
Determination of antibody binding titers by ELISA. The titers of the animal
serum binding antibodies were determined by using an enzyme-linked immu-
nosorbent assay (ELISA) method. In brief, protein or synthesized peptide was
adsorbed onto each well of a Maxisorp high binding plate (Nunc) in PBS, pH 7.4,
overnight at 4°C. For protein ELISA, 100 ng of YU2 or ?V1/2 gp120 protein
expressed from stable Drosophila S2 cells and purified to near homogeneity by
antibody affinity chromatography was adsorbed to each well. The anti-C1/C5
region antibody C11 was used to confirm that similar levels of each protein were
applied to the wells. For peptide ELISA, 40 ng/well of overlapping V1 to V2 loop
peptides or 100 ng/well of V3 peptides was coated onto the ELISA plate surface.
All wells were blocked by blocking buffer containing PBS, 2% dry milk, and 5%
heat-inactivated fetal bovine serum. Serum was serially diluted in blocking buffer
and incubated at RT for 1 h. The wells were washed five times with PBS
containing 0.2% Tween-20, and a secondary anti-rabbit immunoglobulin G
(IgG)-horseradish peroxidase antibody (Jackson Labs) was added to all wells in
PBS–0.2% Tween 20 at a 1:10,000 dilution and incubated for 1 h at RT. After five
washes with PBS–0.2% Tween 20, 100 ?l of the colorometric TMB (3,3?,5,5?-
tetramethylbenzidine) peroxidase enzyme immunoassay substrate (Bio-Rad) was
added to each well, and the reaction was stopped by adding 100 ?l of 1 M H2SO4
to each well. The optical density was read on a microplate reader (Molecular
Devices) at 450 nm, and the end-point titers of the serum antibodies were
defined as the last reciprocal serum dilution at which the optical density signal
was greater than twofold over the signal detected with the preimmune serum.
Primary HIV-1 strains. HIV-1 primary isolates, SF162, 89.6, Bx08, and the
T-cell line-adapted HIV IIIB, were obtained from the National Institutes of
Health AIDS Research and Reference Reagent Program. Viral stocks were
prepared and titrated in phytohemagglutinin and interleukin-2-stimulated hu-
man peripheral blood mononuclear cells (PBMC). The replication-competent
molecular clone BR07 was provided by Dana Gabuzda of the Dana-Farber
Cancer Institute. This virus is a chimeric infectious molecular clone of NL4-3
that contains the nearly full-length env genes from HIV-1 strain BR07 (41). After
initial plasmid transfection of 293T cells, HIV-1 BR07 was expanded in PBMC
as described above.
PBMC neutralization assay. As previously described (37), this assay used
PBMC depleted of CD8 T cells as targets for HIV-1 infection. After CD8
depletion by magnetic beads, the cells were activated with phytohemagglutinin
and interleukin-2. In most cases, the viruses tested were uncloned virus stocks
derived from PBMC cultures, as described above. However, virus YU2 was a
recombinant Env-pseudovirus encoding a green fluorescent protein (GFP) re-
porter gene (37). Virus neutralization was measured using a flow cytometric
single-round infection assay that detects HIV-1-infected T cells by intracellular
staining for HIV-1 p24 Gag antigen or expression of GFP (used for the YU2
pseudotyped virus). For replication-competent viruses, an HIV-1 protease inhib-
itor (indinavir) was used to prevent secondary rounds of virus replication. The
percent neutralization was derived by calculating the reduction in the number of
p24-Ag- or GFP-positive cells in the test wells with immune sera, compared to
the number of HIV-1-positive cells in wells containing preimmune sera from the
corresponding animal. In some cases, serum samples were screened for neutral-
izing activity at a single dilution. All assays included at least two positive control
reagents: an immune globulin from HIV-seropositve donors and an HIV-positive
serum pool. Prebleed values at the equivalent serum dilution were subtracted
before calculating the percentage of postimmune serum HIV-specific neutral-
ization to adjust for nonspecific serum effects on viral entry. To calculate the
dilution of serum that neutralized 50% of infectious virus (IC50), the serum was
serially diluted and the dose-response curve was fit with a nonlinear function
VOL. 80, 2006ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS1415
(four parameter logistic equation) using GraphPad Prism software (San Diego,
Env-pseudotyped virus neutralization and luciferase reporter cell assay. The
assay was performed as previously described (33, 37) with minor modifications as
noted here. Briefly, a HeLa cell line that expresses CD4, CXCR4, and CCR5 was
used as target cells for HIV-1 infection. These cells, initially called JC53-bl and
now termed TZM-bl cells, were obtained from the National Institutes of Health
AIDS Reference and Reagent Repository, as contributed by John Kappes and
Xiaoyun Wu (10, 46, 55). The cells contain Tat-responsive reporter genes for
firefly luciferase and Escherichia coli ?-galactosidase under regulatory control of
an HIV-1 long terminal repeat. In our assays, the level of HIV-1 infection was
quantified by measuring relative light units (RLU) of luminescence that are
directly proportional to the amount of virus input. The assay was performed in
a 96-well microtiter plate using the same format as the PBMC assay described
above, except that 10,000 TZM-bl cells were used in place of PBMC. Approxi-
mately 48 h after virus infection, the cells were lysed, and RLU were measured
using black solid OptiPlates-96F plates (PerkinElmer, Boston, MA) and a Veri-
tas Luminometer (model 1420-061; PerkinElmer) that inject luciferase assay
substrate (Promega) into each well. Pseudoviruses were prepared by cotransfect-
ing 293T cells with an Env expression plasmid containing a full-length gp160 env
gene and an env-deficient HIV-1 backbone vector (pSG3?Env). Pseudotyped
virus-containing culture supernatants were harvested 2 days after transfection,
filtered through 0.45-?m-pore-size filter, and stored at ?80°C. For neutralization
assays, each pseudotyped virus stock was diluted to a level that produced ap-
proximately 100,000 to 500,000 RLU. The cloning and construction of the full-
length gp160 Env expression plasmids used to make the pseudoviruses are
described in detail in a report by Li and colleagues (33). The Env pseudovirus
based on the BR07 viral strain has been previously described (37, 41). Addition-
ally, we isolated two functional Env clones from the DNA of PBMC infected with
the primary isolate BaL. The Env clones BaL.01 and BaL.26 differed at 16 amino
acid positions in gp160 (data not shown).
Peptide inhibition of neutralization. Peptide inhibition neutralization assays
were done in the same assay format as the PBMC fluorescence-activated cell
sorting (FACS)-based neutralization assay or the pseudotype assay, except that
the control or test peptide was added to serum 30 min prior to the addition of
virus. The concentration of peptide reported (usually 15 or 30 ?g/ml) was that
present when peptide, serum, and virus were incubated together as previously
described (22). Control assays demonstrated that the peptide itself did not
significantly affect virus entry. “No peptide” (defined as PBS of equivalent vol-
ume) was used as a negative control, as were several irrelevant peptides as
described below. The effect of the peptide on virus neutralization was reported
as the percent inhibition of neutralization. This was calculated as follows: [(per-
cent neutralization observed with no peptide ? percent neutralization with test
peptide)/percent neutralization with no peptide] ? 100. Several peptides were
used in the competition studies. The YU2 V3 peptide was a 23-mer peptide
(TRPNNNTRKSINIGPGRALYTTG) and synthesized by SynPep (Dublin,
Calif.) (22). As controls, a scrambled V3 peptide (IGPGRATRPNNNFYTTG
TRKSIH) (22) and a corresponding HIV IIIB V3 peptide (24 mer), purchased
from Sigma-Aldrich, were used. The potent anti-V3 monoclonal antibody 447-D
was used as a control to confirm the ability of the V3 peptides and not the
scrambled peptide to inhibit antibody-mediated neutralization as previously de-
scribed (22) (see Fig S3 in the supplemental material). As additional controls, a
mixture of 22 peptides (15 mers overlapping by 9 amino acids spanning the Ebola
[Zaire] viral glycoprotein sequence) was used in some assays to confirm the
specificity of V3 peptide inhibition. For the V1 and V2 region, 15-mer peptides
overlapping by 5 residues were synthesized by New England Peptide, Inc. (Gard-
ner, MA). These included YU2a01 (TDLRNATNTTSSSWE), YU2a02 (SSSW
ETMEKGEIKNC), YU2a03 (EIKNCSFNITTSIRD), YU2a04 (TSIRDKVQK
EYALFY), YU2a05 (YALFYNLDVVPIDNA), YU2a06 (PIDNASYRLISCN
TS), and YU2a07 (SCNTSVITQACPKVS) (see Fig. 5).
Inoculation of guinea pigs with YU2 gp120 and gp140(?/
GCN4) proteins in selected adjuvants. Guinea pigs, four ani-
mals per group (animal 47 in group B died during the course
of the study), were inoculated with either monomeric YU2
gp120 or trimeric YU2 gp140(?/GCN4) protein immunogens
emulsified in Ribi or the GSK adjuvants, AS01B, AS02A, or
AS03. Animals were inoculated a total of five times, with 4- to
7-week intervals between inoculations. Test bleeds were col-
lected 10 days after each inoculation, and the isolated sera
were subjected to ELISA and HIV-1 neutralization assays.
ELISA analysis of the antibodies in the sera elicited by
gp120 and gp140(?/GCN4). After the fourth inoculation, sera
were collected and tested for binding activity to YU2gp120
proteins by ELISA. All groups generated high titers of anti-
gp120 IgG titers, with the end-point titers ranging from 3.2 ?
105to 7.5 ? 106(Table 1), which is consistent with the results
from our previous studies in mice or rabbits (22, 61). Although
most animals generated relatively high antibody titers, the an-
imals from group A (monomeric gp120 in Ribi adjuvant) and
both groups of animals inoculated with either monomer or
trimers in the AS03 adjuvant had slightly lower end-point bind-
ing titers (Table 1). Both the AS01B and AS02A adjuvants
stimulated approximately fivefold higher titers of binding an-
tibodies than Ribi adjuvant in most animals inoculated with
either monomeric or trimeric glycoproteins (Table 1).
Because much of the neutralizing response elicited by YU2
gp120 was directed against the V1 region (see below), we
sought to determine if these elicited binding antibodies were
predominantly elicited against the V1/V2 variable loops. We
first compared the antibody binding titer against gp120 and a
gp120 with the V1/V2 loop deleted (gp120?V1V2). We ob-
served that for the 15 gp120-inoculated animals, 7 exhibited a
fivefold decrease in recognition of gp120?V1V2 compared to
recognition of wild-type gp120 (Table 1, animals 41, 42, 44, 45,
52, 54, and 55 of groups A, B, and C). In the group D animals
(gp120 in the AS03 adjuvant), which had lower titers to gp120,
there was not an observable binding decrease to gp120?V1/V2
proteins. In the animals inoculated with YU2 gp140(?/GCN4)
proteins, 5 out of 16 animals displayed a fivefold decrease in
binding titer to the gp120?V1/V2 proteins compared to wild-
type gp120 (Table 1). These data suggest that although there
are significant levels of binding antibodies directed toward the
V1/V2 region, it is not the single dominant antibody response
elicited by either gp120 or gp140 glycoproteins (Table 1).
Serum ELISA binding activity to V1, V2, and V3 peptides.
As shown below, much of the neutralizing activity elicited by
monomeric YU2 gp120 can be inhibited by a single YU2 V1
peptide but much less so by the YU2 V3 peptide; we sought to
determine how much of the overall binding antibody repertoire
was directed against these variable loop regions. By ELISA, we
tested for the serum binding activity to the pool of V1/V2
peptides, to the single 15-mer V1 a02 peptide, and to the V3
peptide (Table 1 and data not shown). Most of the animal sera
showed high binding end-point titers for the V1 a02 peptide,
ranging from 3.13 ? 105to 1.5 ? 106; the exceptions were that
all the group A animals and animals 59 and 61 had lower
binding titers to this peptide (Table 1). All the animal sera
demonstrated similar binding patterns to the V1/V2 peptide
pool, suggesting that most of the binding antibodies are di-
rected toward the V1 a02 peptide. The binding titers of most of
the sera to the V3 peptide were reasonably high (1.25 ? 104
to 1.5 ? 106) but were less than the levels obtained for binding
to the V1 peptides (Table 1). Taken together, these data sug-
gested that both YU2 gp120 and YU2 gp140(?/GCN4) pro-
teins elicited significant levels of V1- and V3-loop-directed
Trimeric gp140(?/GCN4) proteins elicit more potent and
broader neutralization than monomeric gp120 proteins. After
1416LI ET AL.J. VIROL.
the fourth injection, sera were collected and assayed for neu-
tralization activity in a titration format against the homologous
YU2 virus and the relatively sensitive virus SF162 using a
FACS-based PBMC neutralization assay. We selected sera
from animals from groups D and H that had been inoculated
with monomeric YU2 gp120 or trimeric YU2 gp140(?/GCN4)
proteins in the same AS03 adjuvant. Not all groups were an-
alyzed to conserve limited sera for subsequent analyses. Sera
were tested at an initial dilution of 1:10 and serial fivefold
dilutions. For YU2, at the initial 1:10 serum dilution, the tri-
meric YU2 gp140(?/GCN4) animal sera had a mean (? stan-
dard error of mean) neutralization activity of 89% ? 8.0%,
while monomeric YU2 gp120 animal sera displayed an average
neutralization activity of 56% ? 20% (Fig. 1A). The IC50
values for the reciprocal dilutions of the sera elicited by the
gp140 glycoproteins were at least fivefold higher, ranging from
37 to 186, than the values for monomeric YU2 gp120-elicited
sera, which ranged from 6 to 32. Similarly, for SF162, the sera
from YU2 gp140(?/GCN4) protein-injected animals (group
H) displayed approximately fivefold higher IC50values than the
YU2 gp120 protein-injected animals (group D) (Fig. 1B).
To assess the neutralization breadth of the sera elicited in
selected adjuvants, we tested immune sera obtained after both
the third and fourth inoculations of protein-adjuvant. Sera
were assayed at a 1:5 dilution against the homologous YU2
virus and several heterologous viruses, as shown in Fig. 2.
Within the gp120-adjuvant groups, the percent neutralization
of the YU2 and SF162 isolates generally increased from the
third to the fourth inoculation (Fig. 2). After the third inocu-
lation, sporadic neutralization of IIIB and 89.6 was detected,
but this diminished following the fourth inoculation. Similarly,
there were only two serum samples that possessed greater than
50% neutralization of the Bx08 and BR07 isolates. In general,
sera isolated from the animals inoculated with gp140(?/
GCN4) in the GSK adjuvants displayed more frequent and
more potent neutralization of the YU2, IIIB, and 89.6 isolates
than was achieved by the gp120-inoculated animals (Fig. 2).
From the sera isolated after the third inoculation compared to
after the fourth, increased neutralization of the YU2 and
SF162 isolates was observed, whereas IIIB neutralization di-
minished slightly and 89.6 neutralization diminished dramati-
cally. Sporadic, low-level neutralization of the BR07 and Bx08
TABLE 1. Envelope glycoprotein and V1, V2, and V3 variable loop reactivity of the sera from immunized guinea pigsa
ImmunogenGroup Adjuvant Animal
gp120 gp120?V12 V1V2 pool V1(a02) peptideV3 peptide
aSera were collected after the fourth inoculations, with preimmune sera as a negative control in the assay.
bEnd-point ELISA titers were defined as the last reciprocal serum dilution at which the optical density signal was greater than twofold over the signal detected with
the preimmune serum. Symbols for the end-point titers are as follows: ?/?, 2.5 ? 103; ?, 1.25 ? 104; ??, 6.25 ? 104; ???, 3.13 ? 105; ????, 1.5 ? 106; ?????,
7.5 ? 106.
cND, not determined.
VOL. 80, 2006 ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS 1417
isolates was observed in the gp140-GSK adjuvant groups, with
tralization breadth, albeit of relatively modest potency (Fig. 2).
Assessment of sera neutralization breadth with a recently
standardized HIV-1 assay. We sought to examine the breadth
of neutralization following a fifth inoculation of the gp120 and
gp140(?/GCN4) proteins in the recently standardized assay
designed to permit the cross comparison of neutralization elic-
ited by diverse immunogens (33, 36). Due to the analysis de-
scribed in the present report, there were no sera remaining
from the third and fourth protein inoculations; therefore, we
compared the sera following the second inoculation to that
following the fifth. Initially, we tested the ability of the sera to
neutralize a panel of clade B Env-pseudotyped viruses at a
single dilution of 1:5. As seen in Fig. 3A, the sera from the
gp140(?/GCN4) protein inoculated in the GSK adjuvants ef-
ficiently neutralized YU2, BaL, JR-CSF, and JR-FL deleted of
the 301 glycan (JR-FL?301 ). In general, and as before, the
sera derived from YU2 gp140(?/GCN4) emulsified in the
GSK adjuvants displayed more potent neutralizing activity
than did the sera elicited by gp120 in the GSK adjuvants. The
difference in neutralization between the gp140- and gp120-
elicited sera was statistically significant as determined by a
nonparametric Mann-Whitney test. For most viruses, the P
value derived by this analysis ranges from 0.0001 to 0.056 (see
Fig S1 in the supplemental material).
By comparing the serum neutralization potency from sam-
ples after inoculations 2 and 5, we observed a general increase
in homologous neutralization and, when achieved, heterolo-
gous neutralization generally increased (Fig. 3A). Interest-
ingly, in contrast to this trend, the YU2 gp140(?/GCN4)-
elicited sera after inoculation 2 (and after inoculation 3 in the
previous analysis) neutralized the 89.6 isolate; however, most
of this activity declined following inoculations 4 and 5. After
inoculation 2, all sera elicited by the YU2 gp140(?/GCN4) in
AS02A adjuvant (group G) neutralized the 89.6 virus at values
greater than 80% (Fig. 3A). We assessed the potency of these
sera by serial dilution and 89.6 neutralization to derive IC50
values in excess of 1:640 for the individual serum (see Fig. 6A).
In general, the sera in group G (YU2 gp140 in AS02A) dis-
played the greatest potency, as most individual animal serum
samples from this group could neutralize 7 of the 10 isolates
tested at the level of 50% or greater (Fig. 3A). As before, we
performed a titration of the neutralizing activity against the
homologous YU2 virus from sera elicited in the matched AS03
adjuvant; the gp140(?/GCN4)-inoculated animals displayed
significantly higher neutralization titers with average IC50val-
ues 15-fold higher than the gp120-inoculated animals (1:12 for
the monomer compared to 1:186 for the trimer) (Fig. 3B).
Consistent with the homologous neutralization titers, when the
heterologous virus JR-CSF was tested in the titration assay, a
moderate threefold higher IC50value was observed for the
gp140-GCN4-inoculated animals compared to that for the
gp120-inoculated animals (data not shown). In summary, in both
the PBMC-based assay and the standardized Env-pseudotyped
virus neutralization assay, we confirmed that trimeric gp140(?/
GCN4) proteins elicit more potent and modestly broader neu-
tralization than the monomeric gp120 proteins.
The adjuvants AS01B, AS02A, and AS03 elicit more potent
and broader neutralizing antibodies compared to the matched
immunogen in Ribi adjuvant. By inspection of the neutraliza-
tion data presented in Fig. 2 and 3A, it is apparent that espe-
cially with the gp140(?/GCN4) immunogens, the GSK family
of adjuvants is superior to the commercially available Ribi
adjuvant. After inoculation 4 (Fig. 2), the sera from three out
of four animals inoculated with YU2 gp120 in Ribi adjuvant
could not neutralize either the homologous isolate YU2 or the
lab-adapted isolate IIIB or the sensitive primary isolate SF162
(Fig. 2). From this group, only animal 42 had relatively potent
neutralizing activity against the YU2 and SF162 isolates. The
sera from most animals in all groups injected with monomeric
gp120 in the GSK series of adjuvants had more potent neu-
tralization activity against YU2 and SF162 but less neutraliza-
tion activity against IIIB. The 2 animals in group B (AS01B
adjuvant) were the exception and did neutralize IIIB in the 60
to 70% range at the 1:5 dilution values tested (Fig. 2).
When the mean neutralizing values elicited by the gp140
proteins in each of the four adjuvants were statistically com-
pared by a Mann-Whitney nonparametric analysis, it was ap-
parent that the GSK adjuvants all elicited higher neutralizing
activity at the single dilution point analyzed (Fig. 4). For ani-
mals inoculated with the trimeric gp140(?/GCN4) proteins,
the percentage of homologous YU2 neutralization ranged
FIG. 1. Titration of neutralization activity in sera after four inocu-
lations of YU2 gp120 (group D) or gp140(?/GCN4) (group H) with
the GSK AS03 adjuvant. Sera were tested for neutralization of YU2
(A) and SF162 (B) at decreasing serum concentrations. The average
neutralization values for each group plotted against the serum dilution
factors are shown. Error bars indicate the standard errors of the
1418LI ET AL.J. VIROL.
from 91 to 99% in the AS01B, AS02A, and AS03 adjuvants and
ranged from 0 to 68% in Ribi adjuvant group (P ? 0.03) (Fig.
2 and 4A). For the lab-adapted isolate IIIB and the sensitive
primary isolate SF162, the percent neutralization values
ranged from 50 to 93% in the AS01B, AS02A, and AS03
adjuvants, while it was below 50% in the Ribi adjuvant (group
E) (P ? 0.03) (Fig. 2 and 4B and C). In summary, the GSK
family of adjuvants improves the potency of the neutralizing
antibody response with either the monomeric or trimeric YU2
envelope glycoprotein immunogens compared to the commer-
cially available Ribi adjuvant.
Mapping homologous YU2 neutralization specificity elicited
by monomeric and trimeric immunogens using peptide inhi-
bition of neutralization. To define the neutralization specificity
elicited by monomeric gp120, we performed peptide inhibition
assays. Initially we focused on the V3 region and utilized an
assay that we had established previously (22). We used the
anti-V3 loop monoclonal antibodies 447-D and 2442 to vali-
date the assay, as previously described (22). Briefly, animal
sera were preincubated with synthesized peptides derived from
the YU2 V3 region. In sera, these peptides could potentially
form a complex with the V3 loop-directed antibodies and thus
block the neutralization activity against V3-specific neutraliza-
tion determinants. The percent inhibition of neutralization was
obtained by comparing the percent neutralization in the pres-
ence or absence of peptide to identify V3-directed neutraliza-
tion present in a given serum sample.
For the V3 analysis, serum samples remaining in greatest
abundance after the previous analysis were selected. Ten se-
rum samples from the YU2 gp120-inoculated animals were
analyzed in this experiment. As shown in Table 2, the presence
of V3 peptide in the assay did not greatly inhibit the homolo-
gous YU2 neutralization activity for most of the sera (30% or
less). The exception was the serum from animal 54 in the GSK
AS02A adjuvant group, which showed a 47% activity decrease
following incubation with the V3 peptide. However, many of
the gp120-elicited sera that possessed relatively weak neutral-
ization also displayed some minor fluctuations in neutraliza-
tion activity with and without the V3 peptide. Because of the
increased variability in the assay, and by inspection of the
robust V1 peptide inhibition data below, we interpret the re-
sults to indicate that the homologous neutralization activity
elicited by YU2 gp120 was not predominantly against the V3
When we performed similar V3 peptide inhibition assays
with the more potent YU2 gp140(?/GCN4)-elicited sera, the
FIG. 2. PBMC, FACS-based neutralization assay against five HIV-1 primary isolates and one T-cell-line-adapted isolate, IIIB. Shown are values
from a single-round in vitro neutralization with guinea pig sera after the third and fourth inoculations at a 1:5 dilution against the indicated isolates.
Numbers indicate percent neutralization, as described in Materials and Methods, and are color coded; yellow indicates 50 to 80% neutralization,
and red indicates ?80% neutralization.
VOL. 80, 2006ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS 1419
data clearly demonstrated that little homologous neutralizing
activity was V3 directed (Table 2). The percent inhibition of
neutralization mediated by the V3 peptide was less than 10%
for 11 of 12 animals, and the sera from animals 61 and 63
displayed V3-mediated inhibition levels of 24% and 17% (Ta-
ble 2). These data were consistent with our previous rabbit
immunogenicity, study which demonstrated that the homolo-
gous neutralization activity elicited by trimeric YU2 gp140(?/
GCN4) protein was not predominantly directed toward V3
We then attempted to examine if the specific neutralization
activity was focused on the V1 or V2 loops. To develop a
peptide inhibition assay for this gp120 region, we first selected
two serum samples (animals 44 and 46 from group B) and
preincubated each of the sera with a pool of overlapping 15-
mer peptides derived from the YU2 V1 and V2 loops (Fig. 5A)
(see Materials and Methods). Somewhat surprisingly, we
found that the V1/V2 peptide pool inhibited most of the neu-
tralization activity of these two serum samples. For animal 46,
100% of the neutralization activity was inhibited by the V1/V2
peptide pool (Fig. 5B and Table 2), and for animal 44, 61% of
neutralization was inhibited (Table 2). These results suggested
FIG. 3. (A) Neutralization activity against eight pseudotyped HIV
primary isolates. Shown are values from a single-round in vitro neu-
tralization with guinea pig sera after the second and fifth inoculations
at a 1:5 dilution against the indicated isolates. Numbers indicate per-
cent neutralization, as described in Materials and Methods, and are
color coded; yellow indicates 50 to 80% neutralization, and red indi-
cates ?80% neutralization. ND, not determined. (B) Titration of ho-
mologous neutralization activity in sera after four inoculations of YU2
gp120 (group D) or gp140(?/GCN4) (group H) with GSK AS03. The
average neutralization values plotted against the serum dilution factors
are shown. Error bars indicate the standard errors of the means.
1420 LI ET AL.J. VIROL.
that monomeric YU2 gp120 elicits neutralizing antibodies pre-
dominantly focused on V1 and/or V2 loops. To better define
the specificity, we divided the V1 and V2 peptides into three
test groups, the first containing two peptides, designated V1V2
pool 1. The second group contained a single peptide V1 a02
that we designated pool 2 (we had found that a similar peptide
derived from HXBc2 inhibited HXBc2 homologous neutral-
ization; J. Mascola and G. Nabel, unpublished observations). A
pool of the four most-C-terminal V1/V2 peptides was desig-
nated V1V2 pool 3 (Table 2 and Fig. 5A). We then tested the
inhibition effects of these three peptide sets on 10 selected
animal serum samples. We found that the single peptide V1
a02, derived from the C terminus of V1 loop, inhibited homol-
ogous neutralization of 7 of 10 serum samples at the 100%
level, 2 of 10 serum samples at the 70% level, and 1 serum
sample at the 40% level (Table 2 and Fig. 5B). These results
strongly suggested that the neutralization activity against the
homologous isolate YU2 elicited by monomeric gp120 glyco-
protein was focused to a single epitope within the V1 loop.
Consistent with this observation, most of the other peptides
within the V1 and V2 loop did not show significant inhibition
of neutralization. The one exception was that the serum from
animal 48 was inhibited 35% by the V1V2 pool 1 and 47% by
the V1V2 pool 3 (Table 2). However, this particular serum
sample displayed diverse effects, and the data were difficult to
interpret. By definition, the inhibition of neutralization should
not exceed the 100% level, which was already achieved by the
V1 a02 peptide itself.
To examine if the YU2 gp140(?/GCN4) elicited neutraliza-
tion activity toward V1 and V2 loops, we preincubated the sera
with the three V1/V2 peptide groups. As shown in Table 2, 11
of 12 animal serum samples had less than 20% activity inhibi-
tion by both V1V2 pool 1 and pool 3. Compared to peptide
pools 1 and 3, there was more inhibition of neutralization
inhibited by the peptide a02. Seven of 12 samples had less than
50% inhibition of neutralization by V1 peptide a02, while 5 of
12 samples displayed 50 to 83% inhibition. These data sug-
gested that trimeric YU2 gp140(?/GCN4) elicited neutraliza-
tion activity against the autologous virus YU2 that was par-
tially, but not predominantly, directed toward the V1 and V2
loops, in contrast with neutralizing specificity elicited by mo-
nomeric YU2 gp120. In a similarly designed assay, we also
analyzed selected sera elicited by the gp140(?/GCN4) glyco-
proteins for inhibition by preincubation with a 2F5-binding
peptide. No reduction in homologous neutralization activity by
the 2F5-binding peptide could be observed, although this pep-
tide could efficiently inhibit 2F5-mediated neutralization in the
same experiment (data not shown).
Mapping heterologous neutralization specificity by peptide
inhibition. We observed that although homologous YU2 neu-
tralization and heterologous neutralization of several isolates
increased with repeated inoculations, neutralization of 89.6
declined after inoculations 2 through 5 (see Fig. 2 and 3A).
Less dramatically, neutralization of the IIIB isolate also de-
clined slightly after inoculations 3 to 4 (Fig. 2). To determine
where the initial 89.6 neutralizing activity was directed, we
performed adsorptions of the sera using the YU2 V1/V2 pep-
tide pools and with the V3 peptide prior to performing neu-
tralization assays. As shown in Fig. 6A, when we performed the
adsorption/neutralizations over a range of serum dilutions
from 1:10 to 1:640, most of the neutralization could be re-
moved by the V3 peptide (in this case, a 40% reduction in
neutralization is roughly a threefold difference in viral entry),
but the V1/V2 peptides did not affect neutralization. From
these data, IC50values for 89.6 neutralization could be deter-
mined and were greater than 1:640 for animals 68 to 72. Given
that we could now detect some V3-directed heterologous neu-
tralization, we analyzed selected and relatively potent sera
against other selected isolates. In contrast to 89.6, heterolo-
gous neutralization of JR-CSF elicited by either the trimer or
the monomer in ASO2A adjuvant following the fifth inocula-
tion was not V3 directed (Fig. 6B). We also mapped selected
sera against BaL.01 following the fifth inoculation and found
that similar to 89.6, neutralization was mostly adsorbed by the
YU2 V3 peptide (approximately 10-fold reduction of viral neu-
tralization) (see Fig S3 in the supplemental material), whereas
most JR-FL?301 neutralization following the fifth inoculation
was not V3 directed (data not shown).
FIG. 4. Comparison of gp140-elicited neutralization of the YU2
(A), SF162 (B), and IIIB (C) viruses in the four adjuvants. The hori-
zontal bars indicate the mean neutralization value of each adjuvant
group at a 1:5 serum dilution. For each virus, the Ribi adjuvant group
and the three GSK adjuvant groups together were compared by a
nonparametric Mann-Whitney test.
VOL. 80, 2006 ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS1421
Previously we had observed that emulsified in Ribi adjuvant,
the YU2 gp140(?/GCN4) molecules elicited neutralizing an-
tibodies more efficiently than YU2 gp120 monomers. In that
study, the maximal breadth was observed after repeated pro-
tein boosting without any observed increase in ELISA binding
antibody titers after the inoculation (22). These data suggested
that perhaps repeated boosts either improved the quality or
increased the neutralizing titer. In this study we then sought to
confirm that the gp140 molecules better elicited neutralizing
antibodies in comparison to the monomers with larger num-
bers of animals and in a different species. We also asked if the
adjuvant further improved either the quantitative or qualita-
tive neutralizing responses to the trimeric immunogens. The
YU2 gp120 monomeric proteins were included in parallel as a
comparative control. We focused on a family of proprietary
GSK adjuvants, one of which (AS02A) has shown potent in-
duction of cellular and humoral responses in clinical settings
Here we have shown that the binding titers were slightly but
not substantially increased by emulsification of the YU2 Env
proteins in the GSK adjuvants, but the neutralization potency
was enhanced. Importantly, the YU2 gp140(?/GCN4) trimers
consistently, albeit in some cases modestly, outperform the
YU2 gp120 molecules in terms of both neutralization potency
and breadth, as determined by two independent neutralization
assays, and the values are shown in parentheses and italics.
These data are in agreement with those of our previous studies
(22, 59) and consistent with the data reported in a similar
investigation comparing ADA monomers to trimers by Kim et
al. (27). Mapping of the homologous YU2 neutralization dem-
onstrated that there was a distinct difference in the specificity
of neutralization activity elicited by the YU2 gp120 monomers
compared to the gp140 trimers, indicating that modestly im-
proved neutralization elicited by the gp140 trimers is qualita-
tive and not simply quantitative in nature. Another major find-
ing is that the GSK adjuvants outperform Ribi adjuvant in
terms of neutralization elicited from either the monomeric or
trimeric molecules. Taken together, these two observations
suggest that further modifications of the trimer, in the more
potent GSK adjuvants, may continue to elicit better antibodies
with increased neutralization breadth. There was a trend for a
slight increase in the neutralization breadth of the sera elicited
by the YU2 gp140(?/GCN4) trimers in the AS02A adjuvant in
FIG. 5. YU2 V1 and V2 sequence, peptides, and neutralization specificity mapping. (A) The YU2 V1 sequence is shown in regular type, and
the V2 sequence is in italics. The overlapping V1 and V2 peptides used for inhibition of viral neutralization by the monomer- or trimer-elicited
sera are shown beneath the variable loop sequence. (B) A representative example of peptide inhibition of neutralization assay is shown with sera
from animal 46 after the fourth inoculation at a 1:5 dilution. Peptides were preincubated at selected concentrations with the sera prior to the
neutralization assay as described in Materials and Methods. Peptides are specified below the horizontal axis; PBS indicates no peptide, and the
scrambled V3 and V3 peptides were used as controls.
1422 LI ET AL.J. VIROL.
both the PBMC-based assay and the pseudotype neutralization
assay against IIIB, SF162, and JR-CSF that was marginally
statistically significant (P ? 0.057; data not shown). Further study
of selected adjuvant-protein combinations is certainly warranted.
For some isolates, the neutralizing antibodies elicited by
monomeric gp120 were directed toward the accessible and
immunogenic V3 loop (25, 26, 40, 49, 62). The V2 loop has also
been previously reported as a neutralization determinant (24,
44). Interestingly, for the monomeric YU2 gp120 immunogen,
homologous neutralization mapped to a single region in the C
terminus of the V1 loop. However, based upon our peptide
neutralization inhibition data, the heterologous neutralization
of SF162 and IIIB viruses appeared not to be mediated
through V1-directed antibodies (data not shown). By compar-
ison of homologous V1 residues (using the IIIB molecular
clone HXBc2) coupled with the knowledge that IIIB and
SF162 heterologous neutralization was not V1 directed, it ap-
pears that the YU2 homologous neutralization is likely di-
rected at the N-terminal region of the V1 a02 peptide. More
direct proof of this deduction will require further V1 peptide
fine mapping. Strain-restricted, V1-directed antibodies have
been reported in Abgenix XenoMouse animals inoculated with
SF162 gp120 (23).
The trimer protein elicited homologous neutralizing activity
that is not yet defined: only V3, V1, and 2F5 have been ruled
out (by negative data) from the inhibition of neutralization
assay with the appropriate homologous peptides. Heterolo-
gous neutralization may be likely achieved by some other
means for both the monomer and the trimer. In fact, for a
relatively V3-sensitive molecular clone such as BaL.01, sub-
stantial levels of neutralization elicited by both the monomer
and the trimer were inhibited by the V3-derived peptide. For
less-V3-sensitive strains such as JR-CSF, heterologous neutral-
ization was not V3 mediated (Fig. 6 and Fig. S3B in the sup-
plemental material). By process of elimination and by the
relatively limited neutralization breadth, it is possible that rel-
TABLE 2. Neutralization of YU2 virus and V1, V2, and V3 loop peptide adsorptions of the sera elicited by YU2 gp120 and gp140(?/GCN4)
% Neutralization with peptides (% inhibition)a
V3 peptideV1V2 pool V1V2 pool 1b
V1 a02 V1V2 pool 3c
5757 (0)48 (15)57 (0)0 (100)57 (0)
B AS01B84 ? 6e
82 (2)84 (0)33 (61) 24 (71)
84 ? 8
0 (100)0 (100)
C AS02A 40 (10)
99 ? 2
99 ? 1
FAS01B 99 (0)
77 (22) 94 (5)
aPercent inhibition was calculated by comparing the neutralization activity in the presence and absence of peptides, and the values are shown in parentheses and
bV1V2 pool 1 consists of peptides YU2a01 and YU2a03.
cV1V2 pool 3 consists of peptides YU2a04, -a05, -a06, and -a07.
dAnimal sera that contained a low level of neutralization activity were not analyzed (indicated by underlining). If a value is not shown for neutralization or peptide
inhibition, then the analysis was not determined due to the limitation of available serum.
eFor sera in which the peptide inhibition assays were performed in replicate assays (three or greater), a standard deviation value is shown.
VOL. 80, 2006ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS1423
atively nonpotent CD4 binding site antibodies typified by F105
or b6 (42, 47) or CD4-inducible responses typified by 17b (54)
have been elicited and may mediate neutralization of IIIB.
However, these responses may vary over the course of immu-
nization as the 89.6 isolate, which was not neutralized at all
time points, is weakly neutralized (if at all) by most CD4
binding site antibodies except IgGb12. In fact, 89.6 neutraliza-
tion, when observable, was mostly V3 directed. The waning of
89.6 neutralization (and to a lesser extent the non-V3 neutral-
ization of IIIB) is interesting but potentially disturbing in terms
of boosting selected responses at the cost of others. After two
inoculations, only the trimer-elicited sera could neutralize this
isolate; however, the neutralization waned precipitously be-
tween the inoculations 3 and 4 and was also deficient in the
sera after inoculation 5. This is in contrast to the more potent
and consistent homologous neutralization of YU2 or when
comparing neutralization after inoculations 2 to 5 of the het-
erologous BaL.01, BaL.26, or JR-CSF isolates. For selected
trimer-elicited sera, we mapped the majority of the 89.6 neu-
tralization to the V3 loop. As shown in Fig. S3B, BaL.01
FIG. 6. Peptide neutralization specificity mapping of selected sera against heterologous primary isolates. (A) Percent neutralization of 89.6 is
shown in a bar graph format of individual sera elicited by the gp140(?/GCN4) proteins at the indicated serial dilutions following preincubation
with PBS or peptide in the same volume of PBS. All variable region peptide sequences were derived from YU2, as described in Materials and
Methods. V1V2 pool 1 consists of peptides YU2a01 and -a03. V1V2 pool 3 consists of peptides YU2a04, -a05, -a06, and -a07. (B) Percent
neutralization of JR-CSF following peptide preincubation for the trimer-elicited sera at a 1:10 dilution (left graph) and monomer-elicited sera at
a 1:5 dilution with the exception that a 1:10 dilution was used for sample 54 (right graph).
1424 LI ET AL.J. VIROL.
neutralization also maps to V3 but does not wane; in fact, it
increases with repeated inoculations. Taken together, the data
suggest that initially the elicited V3 loop antibodies are di-
rected against the V3 tip, a region that is conserved between
89.6 and BaL.01. Following inoculations 3 to 4, for reasons that
are not clear the specificity shifts toward tip-flanking residues
of V3, which are not conserved between 89.6 and BaL.01, and
the ability to neutralize 89.6 via V3 antibodies is lost. The more
modest decreases in IIIB neutralization must occur by a dif-
ferent mechanism as this virus appeared not to be neutralized
by YU2-elicited V3 antibodies. This study highlights that even
in sera of modest breadth, there are multiple neutralization
specificities present. The development of tools to map these
diverse specificities is of a high priority, and a systemic ap-
proach to decipher the complex specificities in broadly neu-
tralizing sera by the design of novel adsorption assays and
viruses of defined sensitivity/resistance merits immediate and
further investigation. Since there is evidence and modeling that
V1 and V3 are proximal in the HIV functional spike (6, 28, 31,
34), it is possible that some combination of V1-directed plus
V3-directed neutralizing responses might be elicited and neu-
tralize a greater array of isolates. However, epitopes that
bridge across Env protomers and that are already variable
themselves are likely to be extremely strain restricted. The
recently described trimer-dependent V2- and V3-specific and
strain-restricted 2909 neutralizing antibody may be of this type
In conclusion, the relatively robust homologous neutraliza-
tion elicited by the YU2 gp140 trimers (IC50of 1:186 dilution)
suggests that even for a relatively resistant isolate such as YU2,
there are “chinks in the viral armor.” However, such accessible
regions may be more strain specific, determined by variable
loop interactions (and maintenance of function) and selection
molded by concurrent circulating antibodies (56). This aspect
may be worth further investigation of human sera exhibiting
breadth of neutralization (such analyses are ongoing). These
data emphasize the more general nature of the rare, broadly
neutralizing antibodies (IgGb12, 2G12, 2F5, and 4E10) in
guiding immunogen design, since they recognize more “gen-
eral” chinks in the armor that are displayed by a relatively wide
array of HIV-1 isolates. An obvious follow-up to this study is to
mask (or delete) the V1 region (and perhaps V3) in both the
context of the monomer and especially the trimer to see if the
elicitation of antibodies can be shifted toward the targets im-
plicated in conferring greater neutralization breadth. The gen-
eral strategy of altering glycans to impact on a more focused
antibody response has been suggested in two recent studies
(43, 44). These and other protein modifications will be the
focus of future studies directed toward further improvements
of the gp140 soluble, stable envelope glycoprotein trimeric
We thank Brenda Hartman and Toni Miller for help with the figures
and thank Gary Nabel for helpful discussions. The GSK adjuvants
were obtained via a Materials Cooperative Research and Development
Agreement (MCRADA) between the National Institute of Allergy and
Infectious Diseases (NIAID) and GlaxoSmithKline.
The analysis described in this study was supported in part by the
Intramural Research Program of the Vaccine Research Center, NIAID,
National Institutes of Health, Bethesda, MD.
1. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M.
Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1alpha,
MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Sci-
2. Allan, J. S., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A.
Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major glycoprotein
antigens that induce antibodies in AIDS patients are encoded by HTLV-III.
3. 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-
4. 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.
5. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of
gp41 from the HIV envelope glycoprotein. Cell 89:263–273.
6. Chen, B., E. M. Vogan, H. Gong, J. J. Skehel, D. C. Wiley, and S. C.
Harrison. 2005. Structure of an unliganded simian immunodeficiency virus
gp120 core. Nature 433:834–841.
7. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu,
C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J.
Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate
infection by primary HIV-1 isolates. Cell 85:1135–1148.
8. Dalgleish, A. G., P. C. Beverley, P. R. Clapham, D. H. Crawford, M. F.
Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential
component of the receptor for the AIDS retrovirus. Nature 312:763–767.
9. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di
Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J.
Schall, D. R. Littman, and N. R. Landau. 1996. Identification of a major
co-receptor for primary isolates of HIV-1. Nature 381:661–666.
10. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, X. Wu, W. A. O’Brien, L.
Ratner, J. C. Kappes, G. M. Shaw, and E. Hunter. 2000. Sensitivity of human
immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by
coreceptor specificity defined by the V3 loop of gp120. J. Virol. 74:8358–8367.
11. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M.
Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary
HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5,
CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149–1158.
12. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Na-
gashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A.
Paxton. 1996. HIV-1 entry into CD4? cells is mediated by the chemokine
receptor CC-CKR-5. Nature 381:667–673.
13. Earl, P. L., C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti,
R. W. Doms, and B. Moss. 1994. Native oligomeric human immunodeficiency
virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reac-
tivities. J. Virol. 68:3015–3026.
14. Earl, P. L., R. W. Doms, and B. Moss. 1990. Oligomeric structure of the
human immunodeficiency virus type 1 envelope glycoprotein. Proc. Natl.
Acad. Sci. USA 87:648–652.
15. Earl, P. L., B. Moss, and R. W. Doms. 1991. Folding, interaction with
GRP78-BiP, assembly, and transport of the human immunodeficiency virus
type 1 envelope protein. J. Virol. 65:2047–2055.
16. Earl, P. L., W. Sugiura, D. C. Montefiori, C. C. Broder, S. A. Lee, C. Wild,
J. Lifson, and B. Moss. 2001. Immunogenicity and protective efficacy of oligo-
meric human immunodeficiency virus type 1 gp140. J. Virol. 75:645–653.
17. Evans, J. T., C. W. Cluff, D. A. Johnson, M. J. Lacy, D. H. Persing, and J. R.
Baldridge. 2003. Enhancement of antigen-specific immunity via the TLR4
ligands MPL adjuvant and Ribi. 529. Expert Rev. Vaccines 2:219–229.
18. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry
cofactor: functional cDNA cloning of a seven-transmembrane, G protein-
coupled receptor. Science 272:872–877.
19. Flynn, N. M., D. N. Forthal, C. D. Harro, F. N. Judson, K. H. Mayer, and
M. F. Para. 2005. Placebo-controlled phase 3 trial of a recombinant glyco-
protein 120 vaccine to prevent HIV-1 infection. J. Infect. Dis. 191:654–665.
20. Gao, F., E. A. Weaver, Z. Lu, Y. Li, H. X. Liao, B. Ma, S. M. Alam, R. M.
Scearce, L. L. Sutherland, J. S. Yu, J. M. Decker, G. M. Shaw, D. C.
Montefiori, B. T. Korber, B. H. Hahn, and B. F. Haynes. 2005. Antigenicity
and immunogenicity of a synthetic human immunodeficiency virus type 1
group m consensus envelope glycoprotein. J. Virol. 79:1154–1163.
21. Gorny, M. K., L. Stamatatos, B. Volsky, K. Revesz, C. Williams, X. H. Wang,
S. Cohen, R. Staudinger, and S. Zolla-Pazner. 2005. Identification of a new
quaternary neutralizing epitope on human immunodeficiency virus type 1
virus particles. J. Virol. 79:5232–5237.
VOL. 80, 2006 ANTIBODY RESPONSES ELICITED BY HIV-1 ENV PROTEINS 1425
22. Grundner, C., Y. Li, M. Louder, J. Mascola, X. Yang, J. Sodroski, and R. Download full-text
Wyatt. 2005. Analysis of the neutralizing antibody response elicited in rabbits
by repeated inoculation with trimeric HIV-1 envelope glycoproteins. Virol-
23. He, Y., W. J. Honnen, C. P. Krachmarov, M. Burkhart, S. C. Kayman, J.
Corvalan, and A. Pinter. 2002. Efficient isolation of novel human monoclo-
nal antibodies with neutralizing activity against HIV-1 from transgenic mice
expressing human Ig loci. J. Immunol. 169:595–605.
24. Ho, D. D., M. S. Fung, Y. Z. Cao, X. L. Li, C. Sun, T. W. Chang, and N. C.
Sun. 1991. Another discontinuous epitope on glycoprotein gp120 that is
important in human immunodeficiency virus type 1 neutralization is identi-
fied by a monoclonal antibody. Proc. Natl. Acad. Sci. USA 88:8949–8952.
25. Javaherian, K., A. J. Langlois, G. J. LaRosa, A. T. Profy, D. P. Bolognesi,
W. C. Herlihy, S. D. Putney, and T. J. Matthews. 1990. Broadly neutralizing
antibodies elicited by the hypervariable neutralizing determinant of HIV-1.
26. Javaherian, K., A. J. Langlois, C. McDanal, K. L. Ross, L. I. Eckler, C. L.
Jellis, A. T. Profy, J. R. Rusche, D. P. Bolognesi, S. D. Putney, and T. J.
Matthews. 1989. Principal neutralizing domain of the human immunodeficiency
virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA 86:6768–6772.
27. 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.
28. Kim, Y. B., D. P. Han, C. Cao, and M. W. Cho. 2003. Immunogenicity and
ability of variable loop-deleted human immunodeficiency virus type 1 enve-
lope glycoproteins to elicit neutralizing antibodies. Virology 305:124–137.
29. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T.
Hercend, J. C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule
behaves as the receptor for human retrovirus LAV. Nature 312:767–768.
30. Koch, M., M. Pancera, P. D. Kwong, P. Kolchinsky, C. Grundner, L. Wang,
W. A. Hendrickson, J. Sodroski, and R. Wyatt. 2003. Structure-based, tar-
geted deglycosylation of HIV-1 gp120 and effects on neutralization sensitiv-
ity and antibody recognition. Virology 313:387–400.
31. Kwong, P. D., R. Wyatt, Q. J. Sattentau, J. Sodroski, and W. A. Hendrickson.
2000. Oligomeric modeling and electrostatic analysis of the gp120 envelope
glycoprotein of human immunodeficiency virus. J. Virol. 74:1961–1972.
32. Lalvani, A., P. Moris, G. Voss, A. A. Pathan, K. E. Kester, R. Brookes, E. Lee,
M. Koutsoukos, M. Plebanski, M. Delchambre, K. L. Flanagan, C. Carton,
M. Slaoui, C. Van Hoecke, W. R. Ballou, A. V. Hill, and J. Cohen. 1999.
Potent induction of focused Th1-type cellular and humoral immune re-
sponses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria
vaccine. J. Infect. Dis. 180:1656–1664.
33. 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. Hahn, and D. 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.
34. Losman, B., A. Bolmstedt, K. Schonning, A. Bjorndal, C. Westin, E. M. Fenyo,
and S. Olofsson. 2001. Protection of neutralization epitopes in the V3 loop of
oligomeric human immunodeficiency virus type 1 glycoprotein 120 by N-linked
oligosaccharides in the V1 region. AIDS Res. Hum Retrovir. 17:1067–1076.
35. Lu, M., S. C. Blacklow, and P. S. Kim. 1995. A trimeric structural domain of
the HIV-1 transmembrane glycoprotein. Nat. Struct. Biol. 2:1075–1082.
36. Mascola, J., P. D’Souza, p. Gilbert, B. Hahn, N. L. Haigwood, L. Morris,
C. J. Petropoulos, V. Polonis, M. Sarzotti, and D. Montefiori. 2005. Recom-
mendations for the design and use of standard virus panels to assess neu-
tralizing antibody responses elicited by candidate human immunodeficiency
virus type 1 vaccines. J. Virol. 79:10103–10107.
37. Mascola, J. R., M. K. Louder, C. Winter, R. Prabhakara, S. C. De Rosa, D. C.
Douek, B. J. Hill, D. Gabuzda, and M. Roederer. 2002. Human immunode-
ficiency virus type 1 neutralization measured by flow cytometric quantitation
of single-round infection of primary human T cells. J. Virol. 76:4810–4821.
38. McDougal, J. S., M. S. Kennedy, J. M. Sligh, S. P. Cort, A. Mawle, and J. K.
Nicholson. 1986. Binding of HTLV-III/LAV to T4? T cells by a complex of
the 110K viral protein and the T4 molecule. Science 231:382–385.
39. Miller, S. I., R. K. Ernst, and M. W. Bader. 2005. LPS, TLR4 and infectious
disease diversity. Nat. Rev. Microbiol. 3:36–46.
40. Moore, J. P., Q. J. Sattentau, R. Wyatt, and J. Sodroski. 1994. Probing the
structure of the human immunodeficiency virus surface glycoprotein gp120
with a panel of monoclonal antibodies. J. Virol. 68:469–484.
41. Ohagen, A., A. Devitt, K. J. Kunstman, P. R. Gorry, P. P. Rose, B. Korber,
J. Taylor, R. Levy, R. L. Murphy, S. M. Wolinsky, and D. Gabuzda. 2003.
Genetic and functional analysis of full-length human immunodeficiency virus
type 1 env genes derived from brain and blood of patients with AIDS.
J. Virol. 77:12336–12345.
42. Pantophlet, R., E. Ollmann Saphire, P. Poignard, P. W. Parren, I. A. Wilson,
and D. R. Burton. 2003. Fine mapping of the interaction of neutralizing and
nonneutralizing monoclonal antibodies with the CD4 binding site of human
immunodeficiency virus type 1 gp120. J. Virol. 77:642–658.
43. Pantophlet, R., I. A. Wilson, and D. R. Burton. 2003. Hyperglycosylated
mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120
as novel antigens for HIV vaccine design. J. Virol. 77:5889–5901.
44. Pinter, A., W. J. Honnen, P. D’Agostino, M. K. Gorny, S. Zolla-Pazner, and
S. C. Kayman. 2005. The C108g epitope in the V2 domain of gp120 functions
as a potent neutralization target when introduced into envelope proteins
derived from human immunodeficiency virus type 1 primary isolates. J. Virol.
45. Pinter, A., W. J. Honnen, S. A. Tilley, C. Bona, H. Zaghouani, M. K. Gorny,
and S. Zolla-Pazner. 1989. Oligomeric structure of gp41, the transmembrane
protein of human immunodeficiency virus type 1. J. Virol. 63:2674–2679.
46. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998.
Effects of CCR5 and CD4 cell surface concentrations on infections by mac-
rophagetropic isolates of human immunodeficiency virus type 1. J. Virol.
47. Posner, M. R., T. Hideshima, T. Cannon, M. Mukherjee, K. H. Mayer, and
R. A. Byrn. 1991. An IgG human monoclonal antibody that reacts with
HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. J. Im-
48. Robey, W. G., B. Safai, S. Oroszlan, L. O. Arthur, M. A. Gonda, R. C. Gallo,
and P. J. Fischinger. 1985. Characterization of envelope and core structural
gene products of HTLV-III with sera from AIDS patients. Science 228:593–
49. Rusche, J. R., K. Javaherian, C. McDanal, J. Petro, D. L. Lynn, R. Grimaila,
A. Langlois, R. C. Gallo, L. O. Arthur, P. J. Fischinger, D. P. Bolognesi, S. D.
Putney, and T. Matthews. 1988. Antibodies that inhibit fusion of human
immunodeficiency virus-infected cells bind a 24-amino acid sequence of the
viral envelope, gp120. Proc. Natl. Acad. Sci. USA 85:3198–3202.
50. Sattentau, Q. J., and J. P. Moore. 1991. Conformational changes induced in
the human immunodeficiency virus envelope glycoprotein by soluble CD4
binding. J. Exp. Med. 174:407–415.
51. Sattentau, Q. J., J. P. Moore, F. Vignaux, F. Traincard, and P. Poignard.
1993. Conformational changes induced in the envelope glycoproteins of the
human and simian immunodeficiency viruses by soluble receptor binding.
J. Virol. 67:7383–7393.
52. 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. M. D., J. Donnelly, J. B.
Ulmer, and S. W. Barnett. 2003. Purification, characterization, and immu-
nogenicity of a soluble trimeric envelope protein containing a partial dele-
tion of the V2 loop derived from SF162, an R5-tropic human immunodefi-
ciency virus type 1 isolate. J. Virol. 77:11244–11259.
53. Sun, P., R. Schwenk, K. White, J. A. Stoute, J. Cohen, W. R. Ballou, G. Voss,
K. E. Kester, D. G. Heppner, and U. Krzych. 2003. Protective immunity
induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum
circumsporozoite protein-specific CD4? and CD8? T cells producing IFN-
gamma. J. Immunol. 171:6961–6967.
54. Thali, M., J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and
J. Sodroski. 1993. Characterization of conserved human immunodeficiency
virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.
J. Virol. 67:3978–3988.
55. Wei, X., J. M. Decker, H. Liu, Z. Zhang, R. B. Arani, J. M. Kilby, M. S. Saag,
X. Wu, G. M. Shaw, and J. C. Kappes. 2002. Emergence of resistant human
immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20)
monotherapy. Antimicrob. Agents Chemother. 46:1896–1905.
56. Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-
Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A.
Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutral-
ization and escape by HIV-1. Nature 422:307–312.
57. Weiss, C. D., J. A. Levy, and J. M. White. 1990. Oligomeric organization of
gp120 on infectious human immunodeficiency virus type 1 particles. J. Virol.
58. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley.
1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387:
59. Willey, R. L., R. Byrum, M. Piatak, Y. B. Kim, M. W. Cho, J. L. Rossio, Jr.,
J. Bess Jr., T. Igarashi, Y. Endo, L. O. Arthur, J. D. Lifson, and M. A.
Martin. 2003. Control of viremia and prevention of simian-human immuno-
deficiency virus-induced disease in rhesus macaques immunized with recom-
binant vaccinia viruses plus inactivated simian immunodeficiency virus and
human immunodeficiency virus type 1 particles. J. Virol. 77:1163–1174.
60. 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.
61. 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.
62. Zwart, G., H. Langedijk, L. van der Hoek, J. J. de Jong, T. F. Wolfs, C.
Ramautarsing, M. Bakker, A. de Ronde, and J. Goudsmit. 1991. Immu-
nodominance and antigenic variation of the principal neutralization domain
of HIV-1. Virol. 181:481–489.
1426LI ET AL. J. VIROL.