The Journal of Experimental Medicine
JEM Vol. 201, No. 9, May 2, 2005 1407–1419www.jem.org/cgi/doi/10.1084/jem.20042510
Antigenic conservation and immunogenicity
of the HIV coreceptor binding site
Julie M. Decker,
Michael S. Saag,
James E. Robinson,
David N. Levy,
Cynthia A. Derdeyn,
James A. Hoxie,
and George M. Shaw
Susan Allen, Eric Hunter,
Beatrice H. Hahn,
Peter D. Kwong,
Howard Hughes Medical Institute,
University of Alabama at Birmingham, Birmingham, AL 35294
Institut de Recherche pour le Developpement, University of Montpellier, Montpellier Cedex 5, France
Department of Pathology, Department of Laboratory Medicine, and
Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104
Vaccine Research Center, National Institutes of Health, Bethesda, MD 20892
Department of Pediatrics, Tulane University Health Sciences Center, New Orleans, LA 70112
Department of Medicine,
Department of Microbiology, and
Section of Biostatistics,
International Health, Emory University, Atlanta, GA 30329
Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as
important targets of the adaptive humoral immune response in natural infection and,
potentially, as components of an acquired immune deficiency syndrome vaccine. However,
variability in exposed epitopes and a combination of highly effective envelope-cloaking
strategies have made the identification of such epitopes problematic. Here, we show that
the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and
circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-
induced (CD4i) antibody during natural human infection and that these antibodies bind and
neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of
189 (94%) HIV-1–infected patients had CD4i antibodies that neutralized sCD4-pretreated
HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal
antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and
polyclonal antibodies from HIV-1–infected humans competed specifically with such
monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed
coreceptor binding surfaces were detected in human plasma; these viruses were neutralized
directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate
lentiviruses, functional constraints on receptor binding create opportunities for broad
humoral immune recognition, which in turn serves to constrain the viral quasispecies.
The antibody response to HIV-1 infection is
typically vigorous and sustained, but its effec-
tiveness in virus containment in vivo is un-
certain. We and others have shown in acutely
infected individuals the rapid development of
HIV-1 strain-specific neutralizing antibodies
(Nabs) and the equally rapid emergence of virus
escape mutations (1–4). Such strain-specific
antibody responses are common, and they
clearly drive virus selection in vivo (3, 4).
More broadly reactive Nabs develop over
longer periods (5–7). HIV-1 has evolved a
variety of defense mechanisms to avoid anti-
body recognition, including epitope variation,
oligomeric exclusion, conformational masking,
glycan cloaking, and steric interference at the
virus–cell interface (8–14), and together, they
contribute to virus persistence in the face of an
evolving antibody repertoire (3, 4). But the
precise nature of this evolving antibody re-
sponse in vivo is incompletely understood.
Analysis of HIV-1–specific monoclonal anti-
bodies has revealed variable loop, CD4 binding
site, chemokine coreceptor binding site, surface
glycan, and membrane proximal gp41 domains
as neutralization targets (for reviews see refer-
ences 13, 14), but the prevalence, titers, and
breadth of polyclonal antibody responses to
J.M. Decker and F. Bibollet-Ruche contributed equally to
The online version of this article contains supplemental material.
George M. Shaw:
Abbreviations used: CD4i,
CD4-induced; CRF, circulating
recombinant form; IC
inhibitory concentration; MPER,
region; Nab, neutralizing anti-
body; sCD4, soluble CD4.
ANTIGENIC CONSERVATION OF THE HIV CORECEPTOR BINDING SITE | Decker et al.
these epitopes in humans are generally unknown. This is in
part a consequence of technical difficulty in identifying
epitope-specific neutralizing antibody responses within a
larger context of polyclonal neutralizing and nonneutralizing
antibody reactivities (15–17).
In the present study, we sought to identify immuno-
genic, broadly cross-reactive epitopes on the HIV-1 enve-
lope glycoprotein that might serve as targets of the adaptive
humoral immune response in naturally infected humans. We
hypothesized that conserved requirements for coreceptor
binding among diverse lineages of human or simian immu-
nodeficiency viruses might be reflected in conserved antige-
nicity at the corresponding envelope surface. As a strategy,
we took advantage of the wide evolutionary distance that
exists between HIV-1 and HIV-2 lineages to probe for con-
served neutralization epitopes. The envelope glycoproteins
of HIV-1 and HIV-2 are only
acid sequences (18). As a consequence, they generally ex-
hibit weak antigenic cross-reactivity, and sera from HIV-1–
infected individuals cross-neutralize HIV-2 poorly, if at
all (19–21). Nonetheless, HIV-1 and HIV-2 each require
chemokine coreceptor binding for cell entry, with primary
non–T cell line–adapted viruses of both types generally using
CCR5 (22, 23). Binding of CD4 to HIV-1 gp120 induces
conformational changes in the outer and inner envelope do-
mains, the bridging sheet, and the positioning of variable
loops V1/V2 and V3 (24–30). These changes lead to expo-
sure of the envelope coreceptor binding site, comprised of
the bridging sheet, adjacent surfaces, and possibly the tip of
V3. Antibodies that bind to HIV-1 gp120 preferentially (or
only) after CD4 engagement are referred to as CD4-induced
(CD4i). Typically, these antibodies bind to surfaces that in-
clude or are proximal to the bridging sheet where they com-
pete with coreceptor binding and broadly (but not potently)
neutralize different HIV-1 strains (28–33). Cross-reactivity
between HIV-1–induced CD4i antibodies and HIV-2 has
not been reported. Here, we explore the antigenic cross-
reactivity and inherent immunogenicity of the coreceptor
binding surfaces of HIV-1 and HIV-2 and assess whether
HIV-2, in complex with soluble CD4 (sCD4), might be
useful as a specific probe for HIV-1–elicited, CD4i-neutral-
izing antibodies in humans infected by HIV-1 or immunized
with candidate HIV-1 vaccines.
40% homologous in amino
Plasma from HIV-1–infected patients neutralizes
Table I shows the extent and kinetics of the Nab response to
autologous HIV-1 virus in a patient (133M) after subtype C
HIV-1 infection. Nab titers against the earliest detectable virus
reached 1:2,500 (50% inhibitory concentration [IC
mo of infection and then subsided. Such a response is typical of
patients with newly acquired HIV-1 infection, and it is gener-
ally followed rapidly by virus mutation and escape from neu-
tralization (3, 4). To look for more broadly reactive Nabs in
this subject, we applied these same plasma specimens to the
]) by 11
HIV-2 strain 7312A, a primary CD4-dependent R5 virus (22,
23, 34). As expected, plasma from this HIV-1–infected patient
(133M) exhibited no detectable neutralizing activity against
, a finding consistent with prior studies showing lit-
tle neutralization cross-reactivity between these highly diver-
gent viral lineages (19, 20). However, when HIV-2
pretreated for 1 h with 9nM sCD4 (equal to the IC
virus), the virus became remarkably susceptible to neutraliza-
tion by 133M plasma, with titers of Nab reaching 1:12,500 by
26 mo after infection (Table I). Similar results were obtained in
six additional subjects with primary subtype C HIV-1 infec-
tion, whose Nab titers to sCD4-pretreated HIV-2
from 1:53 to 1:3,361 and peaked between 8 and 24 mo after
acute infection. To determine if the CD4-dependent Nab ac-
tivity that we observed in plasma from subtype C patients was
limited to this virus clade, we studied additional patients chron-
ically infected with HIV-1 subtypes A, B, C, or D. Fig. 1 A
depicts the neutralization profile of plasma from four such pa-
tients against HIV-2
in the absence or presence of sCD4.
In each case, there was a dramatic sCD4-dependent shift of
100–10,000-fold in the susceptibility of HIV-2 to neutraliza-
titers of CD4i Nab titers in these four individuals
ranged from 1:750 to 1:20,000. 15 uninfected normal donors
had no detectable Nabs to HIV-2
with or without sCD4.
HIV-1 CD4i monoclonal antibodies neutralize
If the broadly cross-reactive neutralizing antibody activity that
we observed in HIV-1–infected patient plasma is due to classi-
cal CD4i antibodies, then prototypic CD4i monoclonal anti-
bodies derived from HIV-1–infected patients, which have
been extensively characterized against HIV-1 envelope glyco-
proteins (28–33), might be expected to cross-neutralize HIV-2
in a CD4-dependent fashion. Fig. 1 B demonstrates this to be
the case. Without sCD4, the CD4i monoclonals 17b, 21c, and
plasma specimens from an HIV-1 seroconverter
Neutralization of HIV-1 and HIV-2 by sequential
uncultured month-2 PBMCs and used to prepare pseudotyped virus.
titer of neutralizing antibodies as determined in JC53BL-13 cells
The HIV-1 gp160
gene from patient 133M was PCR amplified and cloned from
JEM VOL. 201, May 2, 2005
19e failed to neutralize HIV-2
dramatic shift in the neutralization curves was observed with all
three antibodies neutralizing HIV-2
is notable that for both the CD4i polyclonal (Fig. 1 A) and
monoclonal (Fig. 1 B) antibodies, the extent of neutralization
90%, and in the case of the clade D plasma
KAMW, 80%. This is due in part to a time- and concentra-
tion-dependent interaction between sCD4 and the gp120 en-
velope glycoprotein because higher sCD4 concentrations and
more prolonged preincubation times (30–120 min) increased
the extent of HIV-2
neutralization by both monoclonal
and polyclonal CD4i antibodies (unpublished data). Steric ac-
cessibility or affinity of CD4i antibodies to their cognate
. In the presence of sCD4, a
potently (Fig. 1 B). It
epitopes may also influence the extent of virus neutralization
because a single mutation (V434M) in the bridging sheet of
, making this amino acid the same as in HIV-1 (see
Site-directed mutagenesis of the HIV-2 bridging sheet alters
HIV-1 CD4i antibody recognition), resulted in a marked shift
of the neutralization curves of 17b and 19e and of three HIV-1
patient plasmas (shown in Fig. 1 C) to the left and downward,
resulting in 100% neutralization of infectious virus.
Multiple primary HIV-2 strains are susceptible to HIV-1
CD4i antibody neutralization
Neutralization of HIV-2 by HIV-1–elicited CD4i antibodies
is not restricted to HIV-2
and derivative strains. HIV-
bodies. Neutralization of HIV-27312A (A and B) and HIV-27312A/V434M (C) in-
fectivity in JC53BL-13 cells (reference 3) was mediated by plasma from
CD4-dependent neutralization of HIV-2 by HIV-1 anti-
patients with HIV-1 clade A (6X4F), B (CUCY2236), C (49M), or D (KAMW)
infection or by the HIV-1 CD4i monoclonal antibodies 21c, 19e, or 17b.
sCD4 concentrations correspond to the IC50 values specific for each virus.
ANTIGENIC CONSERVATION OF THE HIV CORECEPTOR BINDING SITE | Decker et al.
R5-tropic viruses (22, 35), along with five additional primary
HIV-2 patient isolates also demonstrated striking neutraliza-
tion susceptibility to HIV-1–elicited CD4i monoclonal anti-
bodies and to HIV-1–infected patient plasma in patterns that
were similar (but not identical) to HIV-2
, two other well-characterized HIV-2
. Results for
virus was susceptible to 21c and 19e and to a lesser extent
17b, 31H, and ED49. HIV-2
E51 and 31H, but much less susceptible to 17b, compared
. Both viruses were completely resistant to
neutralization by 13 different HIV-1–elicited non-CD4i hu-
are compared in Table II. Each
was more susceptible to
Neutralization titers of HIV-1 monoclonal antibodies and patient plasma against different HIV-2 viruses
sCD49 nM 3 nM15 nM 28 nM6 nM
aValues preceding the slash marks denote the IC50 in ?g/ml for monoclonal antibodies and in reciprocal dilutions for patient plasma specimens, each in the absence of sCD4.
Values following the slash marks denote IC50 values in the presence of sCD4. sCD4 concentrations were adjusted to correspond to the IC50 specific for each virus as indicated in
the bottom row. Dashes denote absent neutralization defined as IC50 ?25 ?g/ml for monoclonal antibodies or ?1:20 for human plasma. Neutralization assays were performed
in JC53BL-13 cells (reference 3).
JEM VOL. 201, May 2, 2005
man monoclonal antibodies, including those targeting the
CD4 binding site (CD4bs), V3 loop, surface glycans, and
gp41. HIV-2UC-1 was also compared with HIV-27312A in its
susceptibility to neutralization by a subset of 10 HIV-1 clade
A, B, C, and D patient plasmas (Table II, bottom). CD4-
dependent Nab titers against HIV-2UC-1 were at least two-
fold higher than for HIV-27312A in two patients (6X4F and
21X0F), threefold lower in one patient (37X4F), and not
substantially different in seven others. For each HIV-1 anti-
body-positive plasma specimen tested, there was a one-to-
three log CD4-dependent shift in the HIV-2UC-1 neutraliza-
tion curve (Table II, bottom).
HIV-1 CD4i antibody binding to HIV-2 glycoprotein
correlates with neutralization
CD4i antibodies in HIV-1 plasma that neutralize HIV-2 in-
fection might also be expected to compete directly with
HIV-1 CD4i monoclonal antibodies for binding to HIV-2
gp120–sCD4 complexes. Fig. 2 shows the results of an assay
using 16 human plasma samples (11 HIV-1 positive; 5 nor-
mal uninfected controls) to compete with biotin-conjugated
19e for binding to HIV-27312A, HIV-2MVP15132, or HIV-1JR-FL
gp120–sCD4 complexes. A mock-treated sample did not
inhibit biotin-labeled 19e binding, which was normalized
to 100%. Unlabeled 19e competed efficiently with biotin-
labeled 19e binding to each of the three HIV glycoproteins.
The five normal control specimens (sample nos. 1–5) showed
no significant competition for biotinylated 19e binding to
any of the three HIV envelope glycoproteins. The 11 HIV-
1–positive patient specimens, however, competed variably
with 19e for binding to both HIV-1 and HIV-2 glycopro-
teins. Sample nos. 13–16 showed the strongest competition
against 19e for HIV-27312A binding, and these samples also
exhibited the highest neutralization titers against HIV-27312A
(reciprocal mean IC50 ? 0.00007 ? 0.00005). Sample nos.
6–9 showed the least competition with 19e for binding
HIV-27312A, and these had the lowest Nab titers against this
virus (IC50 ? 0.023 ? 0.024). Other samples were interme-
diate in binding and neutralization activity. There was a
highly significant correlation between the titers of Nab mea-
sured against HIV-27312A and the efficiency with which these
plasma specimens competed with 19e for HIV-27312A bind-
ing (R2 ? 0.94; r ? 0.97; P ? 0.0001). With the exception
of sample no. 10, the HIV-1–positive patient plasma speci-
mens competed for 19e binding to the HIV-1JR-FL glycopro-
tein more efficiently than to either of the two HIV-2 glyco-
To further examine the correlation between antibody
binding and neutralization, we tested a large number of bi-
otin-labeled HIV-1 CD4i antibodies for binding to HIV-
27312A envelope glycoprotein with and without sCD4. Fig. 3
A shows that the HIV-1–elicited CD4i antibodies that were
found in Table II to neutralize HIV-27312A most efficiently
(19e, 17b, 31H, and 21c) also bound the HIV-27312A gly-
coprotein most efficiently in a CD4-dependent manner,
whereas those antibodies that neutralized poorly, bound
poorly. To further evaluate the breadth of HIV-1 CD4i
monoclonal antibody binding, we tested three antibodies
(19e, 21c, and 17b) for reactivity against additional primate
lentiviruses (Fig. 3 B). The HIV-1 CD4i monoclonal anti-
gp120-sCD4 complexes by human plasma samples from either nor-
mal uninfected donors (sample nos. 1–5) or HIV-1–infected subjects
Blocking of biotinylated 19e binding to HIV-1 and HIV-2 (sample nos. 6–16). Unlabeled 19e effectively competed with biotinyl-
ated (B*) 19e for binding to all gp120-sCD4 complexes and served as a
ANTIGENIC CONSERVATION OF THE HIV CORECEPTOR BINDING SITE | Decker et al.
bodies bound not only HIV-27312A Env–sCD4 complexes,
but also HIV-2CBL20, HIV-2MVP15132, SIVmac239, SIVmne,
and as a control, HIV-1BAL. It is again noteworthy that
gp120–sCD4 complexes from different HIV-2 and SIV strains
were recognized variably by the three HIV-1 CD4i mono-
clonal antibodies, with 19e exhibiting the strongest reactivity
to all viral envelopes, followed by 21c, and then 17b. These
findings, together with the neutralization results, indicate that
the CD4i chemokine receptor binding surfaces of HIV-2
strains 7312A, UC-1, ST/SXB1, CBL20, and MVP15132, as
well as SIVmac239 and SIVmne, all share substantial antigenic
cross-reactivity with each other and with HIV-1.
Site-directed mutagenesis of the HIV-2 bridging sheet alters
HIV-1 CD4i antibody recognition
HIV-2 neutralization by HIV-1 CD4i monoclonal and poly-
clonal antibodies is best explained by antibodies binding to
the conserved chemokine coreceptor binding surface, in-
cluding the bridging sheet. To evaluate this hypothesis di-
rectly, we performed site-directed mutagenesis on the HIV-2
bridging sheet region (36). The primary amino acid sequence
of the bridging sheet of HIV-1 and the corresponding se-
quence of HIV-2 is conserved but not identical (Fig. 4). Sub-
stitutions were made at three positions in the HIV-27312A se-
quence at or near the binding footprints of monoclonals 17b,
21c, and 19e in the corresponding HIV-1 sequence (8, 9,
31). The effects of these mutations on the susceptibility of
the respective viruses to neutralization by HIV-1 monoclonal
and polyclonal antibodies were substantial (Fig. 1 C and Ta-
ble II). Mutations V434M and H419R (HXB2 numbering
system; Fig. 4) made the HIV-2 sequence at these positions
the same as HIV-1, and thus would be expected to enhance
HIV-1 CD4i–antibody binding. The V434M substitution
led to an 80-fold enhancement of 17b neutralization, at least
10-fold enhancement of X5 neutralization, 6-fold increase in
E51 and 31H neutralization, and 2–3-fold enhancement of
ED49 and 19e neutralization. Neutralization enhancement
was not global, however, because there was a concomitant
85-fold decrease in 21c susceptibility and no change in sus-
ceptibility to the HIV-2 monoclonal 1.7A, which binds a
conserved epitope distant from the bridging sheet (Table II).
Similarly, the H419R mutation led to a 2- to 80-fold en-
hancement in neutralization by 17b, 31H, 19e, ED47, and
ED49, but little or no change in susceptibility to E51, 21c, or
HIV-27312A (A) and to additional HIV and SIV (B) gp120-sCD4 com-
plexes. 1.7A is a human HIV-2 gp120-specific monoclonal antibody,
Screening of CD4i monoclonal antibodies for binding to
whereas all other monoclonal antibodies are CD4i antibodies derived from
JEM VOL. 201, May 2, 2005
1.7A. In addition to mutations expected to enhance HIV-1
CD4i antibody binding, we also tested a Q422L mutant,
which had been shown in HIV-1 to reduce CD4i–antibody
binding (e.g., 17b), while allowing the envelope to other-
wise retain its normal receptor binding and entry functions
(31). The Q422L mutation in 7312A resulted in complete
loss of 17b neutralization (?150-fold change), complete loss
in 31H neutralization (?7-fold change), and a 3-fold de-
crease in 21c neutralization, but had little effect on 19e-,
ED49-, or 1.7A-mediated neutralization. Enhanced suscepti-
bility of the V434M and H419R mutants to neutralization
was also observed with most of the HIV-1 patient plasmas
tested (Table II).
Prevalence and titers of CD4i-neutralizing antibodies in
patients infected by diverse HIV-1 subtypes
Plasma samples from 189 individuals infected by HIV-1
clade A, B, C, D, F, G, or H, or by circulating recombi-
nant form (CRF)01, CRF02, or CRF11, were tested for
CD4i Nabs against HIV-2. In preliminary studies, we
tested a subset of 69 of these specimens for reactivity
against the wild-type HIV-2 strain 7312A and its derivative
7312A/V434M. This pilot study showed that the fre-
quency of detection of HIV-2 cross-reactive CD4i Nabs
was modestly higher for the V434M virus (94%) compared
with 7312A (87%). Based on the enhanced sensitivity of
HIV-27312A/V434M, we used this virus to test all 189 patient
plasma specimens for CD4i Nabs (Table III). CD4i Nabs
were detected in 174 (92%) of patients, with median IC50
titers of 0.0004 (1:2,500) and mean titers of 0.004 (1:250).
Titers of CD4i Nab in plasma from clade D and CRF11
patients, considered separately or as a group, were signifi-
cantly greater than for patients in the remaining groups
(P ? 0.0001). We considered the possibility that, despite
the overall similarity in neutralization patterns observed for
the HIV-2 strains depicted in Table II, divergent HIV-2
strains might detect CD4i Nabs in some of the patients’ plas-
mas that tested negative against HIV-27312A/V434M. Thus, we
retested the 15 negative samples, first by Western immuno-
blot to confirm HIV-1 positivity, and then by neutralization
assay against three different HIV-2 strains: UC-1, ST/SXB1,
and 7312A. All 15 samples were Western immunoblot posi-
tive against HIV-1 proteins. Four samples were found to
have CD4i Nabs against one or more of these viruses in titers
ranging from 1:25 to 1:750. Thus, overall, out of 189 HIV-
1–infected patients tested, 178 (94%) had detectable neutral-
izing CD4i antibodies against HIV-2.
Role of CD4i antibodies in natural HIV-1 infection
Previous studies have shown that HIV-1 CD4i antibodies
are largely excluded by steric hindrance from the virus–cell
interface after CD4 engagement, and as a consequence,
CD4i antibodies generally neutralize HIV-1 inefficiently
(12, 28). However, this steric restriction could be overcome
experimentally by using CD4i antibody fragments (Fab or
sFv) or by disassociating (spatially or temporally) envelope–
CD4 engagement from envelope–coreceptor engagement
(12, 28). Given these constraints on CD4i antibody-medi-
ated neutralization, we sought to examine what role CD4i
antibodies might play in vivo. Sodroski et al. (37) first pos-
tulated that CD4i antibodies might constrain virus to CD4
dependence by selecting against envelope mutations that
lead to spontaneous exposure of the viral coreceptor bind-
ing surface (38, 39). Our results support this hypothesis by
showing in naturally infected humans that CD4i antibodies
are prevalent, high-titer, and so broadly cross-reactive that
they neutralize even HIV-2. However, to test more directly
if CD4i antibodies might be active in constraining HIV-1 to
CD4 dependence in vivo, we examined sequential uncul-
tured plasma specimens from four HIV-1–infected patients
(133M, WEAU0575, SUMA0874, BORI0637) for evi-
dence of viruses that contain mutations in envelope that re-
sult in greater spontaneous exposure of the receptor binding
surfaces. 74 full-length, functional gp160 envelope clones
were derived by PCR amplification of plasma virion RNA
and used to pseudotype env-deficient HIV-1 virus for entry
in JC53BL-13 cells. Two clones from patient SUMA0874
(S736-68 and S736-75) were found to be uniquely sensitive
to neutralization by sCD4 (IC50 ?0.05 ?g/ml), indicating
that they might exhibit greater spontaneous exposure of re-
ceptor-binding surfaces than is generally observed in pri-
mary HIV-1 strains (40). These same two clones were also
distinguished from all others that we examined by an isoleu-
cine (I) to threonine (T) substitution at position 309 (HXB2
numbering system) immediately 5? of the GPGR crown of
the V3 loop (Fig. S1, available a http://www.jem.org/cgi/
content/full/jem.20042510/DC1), a position reported by
Quinnan et al. (41) to confer CD4-independent infectivity
and enhanced susceptibility to neutralization in an unrelated
primary HIV-1 strain. We, therefore, first tested clones
S736-68 and S736-75, along with other SUMA clones
lacking the I309T mutation (including S736-68m/TI),
for CD4-independent fusion and infectivity in Cf2Th-
synCCR5 cells, a canine thymocyte cell line that expresses
human CCR5 but lacks CD4 on its surface (42). The S736-
68 and S736-75 envelopes, but not isogenic envelopes lack-
ing the I309T mutation, supported CD4-independent virus
fusion and entry, and this was abolished by treatment with
17b and other HIV-1 CD4i antibodies (unpublished data).
We next tested the S736-68 envelope clone, along with a
site-directed mutant that restored the more common isoleu-
cine at position 309 (S736-68m/TI), for their susceptibility
to sCD4, to an anti-CD4 monoclonal antibody, to the
CD4i monoclonal 17b, and to autologous SUMA plasma in
JC53BL-13 cells (Fig. 5). The S736-68 pseudotyped virus
was far more sensitive compared with the isogenic S736-
68m/TI mutant to neutralization by sCD4, 17b, and autol-
ogous plasma, and it was less sensitive to inhibition by anti-
CD4 antibody. Similar findings were made with S736-75.
These data suggest that the S736-68 and S736-75 envelopes,
like those from some T cell line adapted viruses, have a
spontaneously exposed chemokine coreceptor binding site
ANTIGENIC CONSERVATION OF THE HIV CORECEPTOR BINDING SITE | Decker et al.
SIV (Mac239 and Ver-Tyo1), and HIV-1 (YU2 and HXB2). Bridging
sheet, variable loops, amino acid identities, and site-directed mutations
(H419R, Q422L, and V434M) are indicated. The signal peptide-gp120
cleavage position for HIV-1 is shown. Variable loops (V1/V2, V3, and V4)
have conventionally been defined by disulfide-linked cysteine residues at
their bases as depicted. However, the actual limits of variable loops have
been resolved structurally in the HXB2–CD4–17b crystal complex (refer-
ence 8), and these sequences are indicated by green bars. It is possible that
structural details diverge in the more distantly related HIV/SIV sequences.
The amino acids contributing to the bridging sheet are highlighted in yel-
Envelope gp120 alignments for HIV-2 (7312A and UC1),
low. Blue dots indicate residues contributing to chemokine coreceptor
binding based on site-directed mutagenesis studies (references 29, 30).
Additional amino acids within the stem of V3, including 298R, 301N, 303T,
323I, 325N, 326M, and 327R, may contribute to gp120 interaction with
CCR5 (reference 76). Red dots indicate HIV-1 contact residues for CD4
based on crystal structure analyses (reference 8). Asterisks below the se-
quence indicate conservation of amino acid identity across all five virus
strains. Overall gp120 sequence identity was calculated based on amino
acid residues exclusive of the initiator methionine of the (cleaved) signal
peptide and a gap-stripped alignment of the sequences shown. Except for
SIVverTYO1, sequences were obtained from the HIV Sequence Compen-
JEM VOL. 201, May 2, 2005
and is less dependent on CD4 binding for entry compared
with most primary viruses. Thus, exposure of the corecep-
tor binding surface on primary HIV-1 viral envelopes oc-
curs spontaneously in vivo, but such viruses are exquisitely
sensitive to neutralization by antibodies including those tar-
geting CD4i epitopes.
Breadth of antigenic cross-reactivity in the HIV and SIV
coreceptor binding sites
To examine the breadth of antigenic cross-reactivity in the
coreceptor binding sites of HIV-1, HIV-2, SIVsm, and
SIVagm, we preincubated strains of each virus with CD4i
monoclonal antibodies or plasma from infected subjects
(with and without sCD4) and assayed for virus neutralization
or fusion inhibition. The results showed that natural infec-
tion by these lentiviruses elicits antibodies that neutralize the
homologous virus as well as the evolutionarily divergent vi-
ruses. Fig. S2 (available at http://www.jem.org/cgi/content/
full/jem.20042510/DC1) depicts potent neutralization of the
four viruses by HIV-1–elicited monoclonal and polyclonal
CD4i antibodies. These results extend the findings of Berger
et al., who observed that HIV-1 subtypes A, B, C, D, E, and
F were all susceptible to neutralization by the HIV-1 CD4i
monoclonal antibody 17b (28).
Although much is known about the HIV-1 envelope glyco-
protein (7–17, 24–33), the present study provides new insight
into its immunogenicity and antigenic conservation. Previous
studies suggested that the conformationally dependent core-
ceptor binding surface on HIV-1 was only weakly immuno-
genic and CD4i antibodies were relatively uncommon (31–
33). This paper indicates quite the opposite to be the case.
We find the vast majority (94%) of HIV-1–infected patients
infected by any 1 out of 10 different clades or CRFs harbor
HIV-specific CD4i Nabs with IC50 titers ranging from 1:20
to ?1:100,000. The mean CD4i Nab titer against HIV-
27312A/V434M among 189 subjects was 1:250 and the median ti-
ter was 1:2,500. 114 subjects had Nab titers ?1:1,000, the
highest reaching 1:143,000. In a related study, we found that
8 out of 10 healthy, uninfected human volunteers immu-
nized with ALVAC vCP1452 HIV-1 gp140 alone or in
combination with soluble monomeric HIV-1 gp120 (AIDS-
VAX B/B) developed HIV-1 CD4i-neutralizing antibodies
against HIV-27312A, compared with 0 out of 5 control sub-
jects who were vaccinated with placebo (unpublished data).
To explain the elicitation of CD4i Nabs by soluble HIV-1
gp120 or expressed gp140, we suspect that envelope glyco-
protein is bound to cell surface–associated CD4, undergoes
conformational change, and elicits a CD4i antibody response.
The observation that CD4i antibodies elicited by HIV-1
infection potently neutralized multiple strains of HIV-2 came
as a surprise. Although most primary human and simian len-
tiviruses use CCR5 as a coreceptor for cell attachment and
entry (23), functionally important amino acids in the HIV-1
envelope coreceptor binding region identified by mutagenesis
experiments (8, 29, 30) are only partially conserved in HIV-2,
SIVmac, and SIVagm (Fig. 4). Moreover, conserved receptor
binding would not necessarily be expected to be reflected in
conserved receptor antigenicity (43–45). Thus, the finding
that HIV-1 CD4i monoclonal antibodies such as 19e and 21c
could bind viral glycoproteins as divergent as those from HIV-1,
HIV-2, SIVsm, SIVmac, and SIVmne in a CD4-dependent
fashion (Fig. 3, A and B), and that monoclonal and polyclonal
antibodies from HIV-1–infected humans routinely neutralized
sCD4-triggered HIV-2 (Tables II and III), was quite unex-
pected. We even found that sCD4-treated SIVverTyo1 from
African green monkey (Fig. 4) is susceptible to CD4i neutral-
ization by some HIV-1–infected patient samples in titers as
high as 1:1,000 (Fig. S2). Together, these observations high-
light the extraordinary degree of antigenic conservation linked
to coreceptor binding exhibited by diverse HIV-1 and HIV-2
lineages, and at the same time, an ability of the human hu-
moral immune system to exploit these constraints.
It is of interest to consider the cooperative interactions that
may be occurring among sCD4, the HIV-2 envelope glyco-
protein, and CD4i antibody that result in potent virus neutral-
ization. We have ruled out the possibility that HIV-1–elicited
CD4i antibodies neutralize HIV-2 by binding directly to CD4
because a scorpion toxin-based CD4 mimetic that differs sub-
dium 2002 (reference 18). We determined experimentally the nucleotide
sequence of the SIVverTYO1 clone used in our studies (? phage SAH12)
and found that it differed from the reported sequence of the same clone
in the Compendium at positions 171 (-), 172 (N), 402 (D), 418 (C), and 427
(W). Numbering is according to the HXB2 sequence.
Table III. Prevalence and titers of CD4i-neutralizing antibodies
against HIV-27312A/V434M in plasma of HIV-1–infected subjects
CD4i Nab titersa
SD HIV-1 plasman Positive
Total 189174 (92)0.004 0.00930.0004
aReciprocal IC50 titers of CD4i-neutralizing antibodies against HIV-27312A/V434M pre-
treated with 15 nM sCD4.
ANTIGENIC CONSERVATION OF THE HIV CORECEPTOR BINDING SITE | Decker et al.
stantially in amino acid sequence from CD4 also results in con-
formational changes in HIV-2 gp120 leading to binding and
neutralization by different monoclonal and polyclonal CD4i
antibodies (reference 46 and unpublished data). If sCD4 does
not interact directly with CD4i antibodies, then it must en-
hance the susceptibility of virus to neutralization by inducing
conformational change and exposure of CD4i epitopes, but in
a cooperative manner because the magnitude of HIV-2 neu-
tralization we observe is far greater than would be expected on
the basis of additive stoichiometry. Of note, Berger et al. (47)
have demonstrated cooperative interactions between different
gp120 protomers within a trimer complex of HIV-1.
A role for CD4i antibodies in natural HIV-1 infection
may become apparent. Our data, together with other results
(37, 41), suggest that HIV-1 variants with exposed corecep-
tor binding surfaces and varying degrees of CD4 indepen-
dence, are generated spontaneously in vivo where they are
almost certainly neutralized by CD4i or other HIV-1–spe-
cific antibodies. In fact, four studies have now shown that
single amino acid substitutions in the HIV-1 glycoprotein,
either at the base of V1/V2 (3, 48) or in the V3 loop (the
present text and reference 41), are sufficient to confer on the
virus varying degrees of CD4 independence, spontaneous ex-
posure of the coreceptor binding site, and enhanced suscepti-
bility to CD4i Nabs. Principles of viral dynamics suggest that
such mutations must be occurring in vivo on a virtually con-
tinuous basis, as has been documented for comparable muta-
tions leading to antiretroviral drug resistance (49). Thus,
CD4i antibodies may influence HIV-1 natural history and
pathogenesis to a greater extent than is currently recognized
by constraining virus to CD4 dependence. Consistent with
this interpretation, Gabuzda et al. have shown that HIV-1 vi-
rus within the central nervous system (where circulating anti-
bodies are relatively excluded) has less dependence on cell
surface–bound CD4 for attachment and entry (50). CD4i an-
tibodies could also influence the frequency of R5/X4 core-
ceptor switching (51) and target viruses with short or other-
wise constrained envelope variable loop sequences (52).
The discovery that sCD4-triggered HIV-2 is susceptible
to binding and neutralization by HIV-1–elicited CD4i anti-
bodies has practical application in studies of HIV-1 natural
history and vaccine assessment. A number of investigative
groups have attempted to stabilize the HIV-1 envelope gly-
coprotein in a CD4-bound configuration to use it as an im-
munogen designed to elicit antibodies against viral receptor
surfaces or other intermediate envelope structures (53–55).
But methods to selectively identify and titer Nabs specific for
such epitopes have been limited. Here, we show that neu-
tralization of sCD4-treated HIV-2 represents an extremely
sensitive and specific assay to detect HIV-1–elicited CD4i
antibodies. Investigators have also targeted the membrane-
proximal external region (MPER) of HIV-1 gp41 for vac-
cine development (56–66) because conserved epitopes in
this region are capable of eliciting broadly reactive Nabs in
natural infection (56–58, 65). But again, neutralization assays
are lacking that allow for the sensitive and specific detection
of MPER epitope-specific Nabs (17). We considered the
possibility that HIV-2 could act as a “molecular scaffold” on
which to present these and other HIV-1 epitope-specific an-
tigens in the context of a functional envelope glycoprotein
that does not otherwise cross-react with HIV-1–neutralizing
antibodies. In recent studies, we have identified and modi-
in JC53BL-13 cells (reference 3) by sCD4 (A), anti-CD4 monoclonal
antibody RPA-T4 (B), CD4i monoclonal antibody 17b (C), and autolo-
gous patient plasma from day 278 after acute infection by HIV-1 (D).
Neutralization of S736-68 and S736-68m/TI infectivity
JEM VOL. 201, May 2, 2005
fied by site-directed mutagenesis HIV-2 strains that can be
used to detect and titer neutralization by the HIV-1 gp41
MPER-specific human monoclonal antibodies 4E10 and
2F5 with high sensitivity and specificity (unpublished data).
Thus, the strategy described in this paper of using HIV-2 en-
velope glycoproteins in the context of infectious virions or
as isolated proteins to detect HIV-1 epitope-specific neutral-
izing antibodies may find application in the assessment of
candidate vaccines and in studies of HIV-1 natural history.
MATERIALS AND METHODS
Plasma specimens. Pre-existing coded plasma samples from 189 HIV-1–
infected subjects and 15 uninfected normal control individuals were ana-
lyzed. Human subjects gave informed consent and protocols received (Uni-
versity of Alabama at Birmingham) institutional review board approvals.
Cell entry and neutralization assays. Plasma samples and monoclonal
antibodies were assayed for Nab activity using a modification of recently de-
scribed HIV entry and fusion assays (3, 32, 67). These assay systems employ
the HeLa cell-derived JC53BL-13 cell line (National Institutes of Health
AIDS Research and Reference Reagent Program catalogue no. 8129,
TZM-b1), which has been genetically modified to constitutively express
CD4, CCR5, and CXCR4, and the canine thymocyte cell line Cf2Th-
synCCR5, which expresses human CCR5 but not CD4 (42).
Virus stocks. HIV-2 proviral clones pJK7312A (GenBank/EMBL/DDBJ
accession no. L36874), pJK7312A/V434M, pJK7312A/H419R, pJK7312A/
Q422L, and pJSP4-27 (ST/SXB1; references 22, 68–70) were used to
transfect 293T cells. HIV-2 UC-1 env (22, 35) and HIV-1 133M env,
cloned in pSM and pCR3.1, respectively, were cotransfected with
pSG3deltaEnv or pJK7312AdeltaEnv to create infectious pseudovirions, as
described previously (3). HIV-1 env genes cloned in pcDNA3.1 were
cotransfected with an HIV-1 reporter virus (pNLENG1-ES-IRES) contain-
ing an enhanced green fluorescence gene (67) for virus entry studies in
Binding and competition assays. Biotinylated monoclonal antibodies
(31–33, 71–76) were tested for binding to HIV-2, SIV, or HIV-1 gp120
envelope glycoproteins (34, 35, 68–70, 77–79) captured on microtiter plates
coated with mAb 2.6C or EH21, as previously described (31, 32). Before
the addition of biotin-labeled antibodies, gp120 was preincubated with
1–10 ?g/ml sCD4 (R&D Systems) or a mock preparation and with or
without competing plasma specimens.
Monoclonal antibodies. mAbs are described in supplemental Materials and
methods (available at http://www.jem.org/cgi/content/full/jem.20042510/DC1).
Molecular cloning, sequencing, and mutagenesis. Full-length gp160
envelope genes were amplified by nested PCR from plasma HIV-1 RNA,
cloned, and sequenced as previously described (3, 49). Sequences are depos-
ited in GenBank/EMBL/DDBJ (accession nos. AY223761-90, AY223720-
Statistical analyses. Linear regression, Pearson correlations, Fisher’s exact
test, and Wilcoxon rank sum test were performed on primary and log trans-
formed datasets. Calculations were performed in SAS.
Online supplemental material. Fig. S1 shows the complete amino acid
sequences for 31 gp160 envelope clones derived from plasma virus from
subject SUMA0874. Four additional gp160 sequences corresponding to
site-directed mutants of wild-type clones S736-68 and S736-73 containing
substitutions at positions 308 or 309 (HXB2 numbering system) are desig-
nated S736-68m/TI, S736-68m/PI, S736-73m/TT, and S736-73m/PI.
Fig. S2 depicts sCD4-dependent neutralization of different HIV and SIV vi-
ruses by HIV-1–elicited CD4i antibodies. Included in supplemental Materi-
als and methods are detailed descriptions of all materials and methods. On-
line supplemental material is available at http://www.jem.org/cgi/content/
We thank the participants and staff of the Birmingham, Lusaka, and Kigali HIV study
sites; D. Burton for providing monoclonal antibody reagents; S. Hu for providing
purified SIVmne gp160; C. Weiss for providing pUC-1env; G. Air and S. Soong for
helpful discussions; and W. Abbott for artwork and technical assistance.
This work was supported by the National Institute for Allergy and Infectious
Diseases/National Institutes of Health (NIH) Acute Infection and Early Disease
Research Program initiative (no. AI41530), the UAB Center for AIDS Research (no.
AI27767), and grants from the NIH (nos. AI35467, AI24030). We also thank Bristol-
Meyers Squibb, Glaxo-Smith-Kline-Agouron, and Merck for ongoing support of
acute HIV-1 infection studies.
The authors have no conflicting financial interests.
Submitted: 9 December 2004
Accepted: 11 March 2005
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