JOURNAL OF VIROLOGY, May 2009, p. 5077–5086
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 10
Enhanced Exposure of the CD4-Binding Site to Neutralizing
Antibodies by Structural Design of a Membrane-Anchored
Human Immunodeficiency Virus Type 1 gp120 Domain?
Lan Wu,1Tongqing Zhou,1Zhi-yong Yang,1Krisha Svehla,1Sijy O’Dell,1Mark K. Louder,1
Ling Xu,1John R. Mascola,1Dennis R. Burton,2James A. Hoxie,3Robert W. Doms,4
Peter D. Kwong,1and Gary J. Nabel1*
Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, 40 Convent Drive, Bethesda, Maryland 20892-30051;
Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-2, La Jolla,
California 920372; Department of Medicine, University of Pennsylvania, 356 Biomedical Research Bldg. II/III,
421 Curie Boulevard, Philadelphia, Pennsylvania 191043; and Department of Microbiology, University of Pennsylvania,
Room 225 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, Pennsylvania 19104-60764
Received 16 December 2008/Accepted 24 February 2009
The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved
surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env)
glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site
(CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which
was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252
to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env
spike as measured by flow cytometry. Truncation of the ?20-?21 hairpin (residues 422 to 436 to Gly-Gly)
improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3
loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface
calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD
increased to over 30%. This OD variant [OD(GSL)(??20-21)(hCD4-TM)] was recognized by b12 and another
CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency.
Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex
antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or
to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.
The human immunodeficiency virus type 1 (HIV-1) enve-
lope is composed of surface gp120 and transmembrane gp41.
Initial attempts to develop HIV vaccines through the induction
of antibodies focused on recombinant gp120 glycoproteins.
Two phase III clinical trials conducted in the United States and
Thailand showed no protection from a gp120-based subunit
vaccine against HIV infection, nor did the vaccine delay HIV-1
disease progression (11, 25). In addition, a phase II trial com-
pleted in Thailand with a live recombinant HIV-1 canarypox
vaccine (vCP1452) in combination with a gp120 subunit pro-
tein did not stimulate a markedly improved immune response
(28). The lack of efficacy was likely to be related to its failure
to elicit broadly neutralizing antibodies (4, 10, 33).
Several broadly neutralizing human monoclonal antibodies
(MAbs) have been derived from infected individuals, including
immunoglobulin G1 (IgG1) b12, 2G12, 2F5 and 4E10, which
are directed against CD4-binding-site (BS), carbohydrate, and
membrane-proximal regions of HIV Env (reviewed in refer-
ence 9). Among the most potent, the b12 antibody occludes the
site of CD4 binding on gp120 and prevents virus attachment to
CD4 on target cells (39). Other CD4-BS antibodies recognize
epitopes on monomeric gp120 that overlap with b12 but lack
the ability of b12 to neutralize primary HIV-1 isolates (5). An
understanding of the specificity of b12 binding, neutralization,
and protection should aid in the development of immunogens
that induce neutralizing antibodies of a similar specificity.
The structure of the b12-gp120 complex (39) shows that b12
binds to a conformationally conserved surface, which is cen-
tered around the CD4-binding loop on the outer domain of
gp120. In the CD4-bound conformation of gp120, the CD4-
binding loop or ?15-strand makes antiparallel intermolecular
hydrogen bonds to the C? strand of CD4 (14). Overall, the
outer domain of gp120 comprises 82% of the gp120 contact
surface with b12, while most of the contacts outside of the
outer domain have marginal importance (39). One exception,
however, are contacts to the loop connecting the outer domain
with the ?5-helix of the inner domain (39), which appear to be
Because it represents the smallest structural unit containing
the b12 epitope, and therefore maximizes the b12-immuno-
genic surface relative to the overall surface, an outer domain-
only immunogen with high b12 affinity represents an attractive
immunogen. An outer domain construct (named OD1) was
previously derived from HIV-1 strain YU2 gp120 and found to
bind 2G12 and a number of anti-V3 antibodies (36); however,
b12 binding to this construct was difficult to detect by enzyme-
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-
3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: (301)
496-1852. Fax: (301) 480-0274. E-mail: firstname.lastname@example.org.
?Published ahead of print on 4 March 2009.
linked immunosorbent assay, probably due to an enhanced off
rate (36, 39). A large, relatively flat interface exists between the
inner and outer domains of gp120 in both CD4-bound and
b12-bound conformations. We reasoned that the removal of
the inner domain might partially destabilize it and decided to
replace the inner domain with another polar surface, the cell
membrane. We expressed outer domain proteins (ODs) in
various membrane-anchored forms and tested their abilities to
bind b12. An HIV-1 clade B R5 and X4 dual-tropic virus, R3A,
was selected as a prototype (20). Laboratory-adapted virus
strain R3A TA1 contains a truncated V1/V2 and a truncated
V3 (named 9,9), maintains CCR5 tropism, and is highly sen-
sitive to b12 neutralization (15, 23). We used available atomic-
level structures to model an R3A gp120 core and to design
truncations of flexible, potentially immunodominant struc-
tures, which emanate from OD, including the ?20-?21 hairpin
and the V3 loop. Thus, by using structure-based design to
modify the OD form of R3A TA1, we attempted to remove
strain-specific determinants, to enhance cell-surface expres-
sion, and to increase specific b12 binding compared to other
MATERIALS AND METHODS
Cell lines, media, and antibodies. 293F and 293T human embryonic kidney cell
lines were purchased from Invitrogen (Carlsbad, CA) and were maintained in
Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine
serum (Sigma, St. Louis, MO). Human IgG1 isotype control was obtained from
Alexis Biochemicals (San Diego, CA). MAbs b12 and b13 were produced in 293F
cells and purified with a protein I affinity column. CD4 was obtained from the
NIH AIDS Research and Reference Reagent Program. 17b, 15e, and F91 were
kindly provided by James Robinson (12, 14, 21, 24, 29). b3, b6, and b11 were
generated as described previously (1, 3, 24, 27, 29). m14 and m18 were prepared
by Dimiter S. Dimitrov (29, 37, 38). F105 was made by Marshall R. Posner (24,
26, 29, 31), and 2G12 was kindly provided by Hermann Katinger (39). fluorescein
isothiocyanate-conjugated Affinipure goat anti-human IgG(H?L) was purchased
from Jackson ImmunoResearch (West Grove, PA). Human sera and plasma
samples were described previously (17).
Outer domain genes. gp145?CFI?V12(9,9) was derived from the HIV-1 R3A
TA1 envelope (15, 23) with cytoplasmic, cleavage-site, fusion peptide, interheli-
cal-domain, and V1V2 deletions, as previously described (7, 34). The outer
domain construct OD(9,9)(hCD4-TM) was made by PCR of amino acids 252 to
482 (252PVVST……WRSE482) (numbering based on HXBc2) from R3A-
gp145?CFI?V12(9,9). This fragment was fused to the mouse interleukin-2
(IL-2) signal peptide sequence (MYSMQLASCVTLTLVLLVNSGPR) and the
human CD4 transmembrane domain (GSGSLIVLGGVAGLLLFIGLGI). V3
derivatives of the same construct and ?20-21 replacement with Gly-Gly were
made using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The
TABLE 1. Neutralization (50% inhibitory concentration) by antiserum from HIV-positive patient serum Zeptometrix 1642 against HIV-1
isolates of diverse clade origin
Beijing, People’s Republic of China
Beijing, People’s Republic of China
Beijing, People’s Republic of China
Beijing, People’s Republic of China
aTCLA, T-cell line adapted; ccPBMC, cocultured peripheral blood mononuclear cells; ucPBMC, uncultured peripheral blood mononuclear cells.
5078WU ET AL.J. VIROL.
corresponding D368R mutants were also generated using the QuikChange mu-
tagenesis kit. All envelope constructs were expressed under the cytomegalovirus
(CMV) promoter in the CMV/R vector (34).
Structure-based OD design and surface area calculations. An R3A gp120 was
modeled from the structure of the gp120 core with V3 (Protein Data Bank
accession number 2B4C) (13) using SwissModel (http://swissmodel.expasy.org
/SWISS-MODEL.html) in alignment mode, while the target sequence of the
R3A core with V3 was aligned to the JR-FL sequence. Variants of OD contain-
ing the primary binding site for b12 were visualized with the program XFIT as
provided in the XtalView package (19). Truncations of the ?20-?21 hairpin were
designed, which retained OD-hydrogen bonding while removing areas of struc-
tural differences between the CD4- and b12-bound structures of core gp120
(Protein Data Bank accession numbers 2NXY and 2NY7) (39). Truncations of
the V3 loop were designed to retain hydrogen bonding at the V3 base while
providing variation in the connecting loop. Variants included Ser-Gly minimal
linkers, basic and acidic linkers, and linkers containing N-linked glycosylation.
Surface areas were calculated with the program GRASP (22).
Surface staining of ODs. 293T cells were transfected with plasmids expressing
OD constructs using the ProFection mammalian calcium phosphate transfection
system (Promega, Madison, WI) according to the manufacturer’s instructions.
Twenty-four hours after transfection, cells were collected with phosphate-buff-
ered saline (PBS) containing 2 mM EDTA and washed with PBS. Cells were
stained with the indicated antibodies (5 ?g/ml), human serum, or plasma samples
(1:100 dilutions) for 30 min on ice. Cells were then washed twice with PBS and
incubated with fluorescein isothiocyanate-conjugated Affinipure goat anti-hu-
man IgG(H?L) (5 ?g/ml) for 30 min on ice and then washed with PBS and
resuspended in PBS with 0.5% paraformaldehyde. Samples were run on a BD
FACSCalibur (see Fig. 1 and 5) or BD LSR-II (see Fig. 2 to 4 and 6) flow
cytometer using FACSDiva software (BD Biosciences, San Jose, CA) and ana-
lyzed with FlowJo 8.6.1 software (Tree Star, Ashland, OR).
Human plasma absorption and neutralization assay. 293F cells were trans-
fected with OD(GSL)(??20-21)(hCD4-TM) or OD(GSL)(??20-21)(hCD4-
TM)(D368R) plasmids using 293fectin (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instructions. Cells were harvested 36 to 48 h after transfec-
tion, washed with PBS, and incubated with human plasma Zeptometrix 1642 (5 ?
107cells/50 ?l plasma in 500 ?l PBS) or MAb b12 (1 ? 109cells/10 ?g MAb in
1000 ?l PBS) on ice for 30 min. The absorbed supernatants were harvested and
filtered for cell surface staining or neutralization assays.
Neutralization was performed using a pseudovirus-based single-cycle infectiv-
ity assay using TZM-bl cells as described previously (17). The results are re-
ported as the 50% inhibitory concentration (the plasma dilution producing 50%
virus neutralization) in Table 1. In the absorbed plasma neutralization assay,
dilutions of absorbed plasma or MAb were used, and the percent neutralization
was calculated compared to that of controls with no plasma added.
Serum or plasma samples from patients 1, 20, 45, US21, and Zeptometrix 1642
all displayed moderate to potent neutralizing activity against diverse strains of
HIV-1. Sera/plasma from patients 1, 20, 45 and US21 were derived from clini-
cally symptomatic HIV-1-infected subjects who were not on antiretroviral ther-
apy. Zeptometrix 1642 was a gift from David Montefiori (Duke University) and
was initially obtained from the Zeptometrix Corporation (Buffalo, NY). There
was no clinical information available on this plasma. Sera/plasma samples from
patients 1, 20, and 45 were provided by Mark Connors, NIAID (17, 18).
Binding of transmembrane forms of the OD glycoprotein to
the b12 antibody. To evaluate the reactivity of R3A TA1 en-
velope derivatives, several deletion mutations, including the
elimination of the V1 and V2 regions, truncation of V3 leaving
9 amino acids on each side of the stem V3(9,9), and deletion of
the cleavage, fusion, and interhelical (?CFI) domains, were
prepared in a gp145 form of this Env to generate gp145
?CFI?V12(9,9) (Fig. 1A) (7, 15, 23). When expressed on
transfected 293T cells, this glycoprotein was able to bind to the
broadly neutralizing MAbs b12 and 2G12 (Fig. 1B). A negative
control vector was created with a mutation (D368R) in the
?15-strand, a portion of gp120 which is critical for its interac-
tion with CD4 (14). gp145?CFI?V12(9,9) (D368R) showed no
binding to b12, whereas 2G12 binding was unaffected (Fig. 1B)
(2G12 recognizes a cluster of ?1-2 mannose residues on the
silent face of the outer domain of gp120  and whose bind-
ing is independent of b12). A membrane-anchored OD variant
was generated from R3A TA1 gp145?CFI?V12(9,9) by ex-
pressing gp120 residues 252 to 482 with the IL-2 signal peptide
sequence and human CD4 transmembrane domain [OD(9,9)
(hCD4-TM)] (Fig. 2A to D and F). This variant of gp120 re-
and F). Residues 252 and 482 are spatially close in unliganded,
FIG. 1. R3A gp145?CFI?V12(9,9) can bind b12, and binding is eliminated by the D368R mutation. (A) Schematic representation of functional
domains and mutations in the HIV-1 R3A Env glycoprotein gp145?CFI?V12(9,9). Deletions include the V1-V2 loop, the tip of the V3 loop, the
gp120/gp41 cleavage site and fusion domain, and the region between the two heptad repeats. (B) MAb b12 (gray lines) or 2G12 (solid black lines)
or human control IgG (dashed lines) binding to 293T cells transfected with a CMV/R vector expressing gp145?CFI?V12(9,9) or
gp145?CFI?V12(9,9)(D368R) was analyzed by flow cytometry.
VOL. 83, 2009TRANSMEMBRANE HIV gp120 OD BINDS b12 ANTIBODY5079
CD4-bound, and b12-bound forms of gp120, and charged resi-
dues at position 252 and 482 were retained to define the mem-
brane boundary. In addition, the truncation and membrane an-
chor were designed to place the membrane into a position similar
to one that the native viral spike is expected to occupy.
OD(9,9)(hCD4-TM) exhibited b12 and 2G12 binding when ex-
pressed on 293T cells by flow cytometry similarly to the
gp145?CFI version (Fig. 2G, left, versus Fig. 1B, middle).
Cell Number (%Max)
FIG. 2. R3A outer domain construct containing the human CD4 transmembrane recognizes b12. (A) Structure of core gp120 in CD4-bound
and b12-bound contexts. The gp120 core structure is shown in ribbon diagram format with the surface corresponding to the b12 epitope highlighted
in blue. (B) Domain separation. Residues 252 to 482, which correspond roughly to the outer domain of gp120 with the ?5-helix, are depicted
separate from the rest of the inner domain. (C) Outer domain with the ?5-helix, rotated 90° from that shown in B, with the surface colored blue
for b12 epitope and otherwise red for the outer domain and gray for the ?5-helix. (D) Same as C but colored according to the chemistry of the
underlying amino acids (red, negatively charged; blue, positively charged; white, apolar). (E) Outer domain with the ?5-helix, with transmembrane
linker shown with membrane and N-linked glycan. C-term, C terminus. (F) Schematic representation of the outer domain construct
OD(9,9)(hCD4-TM). The construct is from R3A gp145?CFI?V12(9,9) residues 252 to 482 (based on the amino acid residue numbers of HXB2)
with an IL-2 leader sequence and the hCD4 transmembrane domain. The ?20-?21 loop deletion construct is shown below. (G) MAb b12 (blue)
or 2G12 (green) or human control IgG (red) binding to 293T cells transfected with OD(9,9)(hCD4-TM) or OD(9,9)(??20-21)(hCD4-TM) was
analyzed by flow cytometry.
5080 WU ET AL.J. VIROL.
The ?20-?21 region of gp120 differs significantly among
unliganded, b12-bound, and CD4-bound conformations of
gp120 (8, 14, 39). In the CD4-bound conformation, ?20-?21
forms half of a well-ordered four-stranded sheet, named the
bridging sheet, which contains elements critical for both CD4
and CCR5 binding (14). Meanwhile, in unliganded and b12-
bound conformations, ?20-?21 forms a flexible hairpin (8, 39).
A truncation of ?20-?21 between residues 424 and 432 was
previously observed to increase b12 binding in strains derived
from HIV-1 clade B virus IIIBand SHIV-89.6 (2). To deter-
mine whether an analogous truncation in an OD expression
vector could similarly increase b12 reactivity, a Gly-Gly dipep-
tide replacement of ?20-?21 residues between residues 422
and 436 was introduced into OD-transmembrane (OD-TM)
vectors. While the introduction of this deletion increased re-
activity with b12, binding to the control 2G12 MAb also im-
proved (Fig. 2B, right), suggesting that the ?20-?21 truncation
improved the overall protein expression of OD and did not
selectively enhance the exposure of the site of b12 binding.
Selective modification of the V3 base after V3 truncation
increased b12 reactivity with OD-TMs. The structure of a
V3-containing gp120 core reveals that the V3 is exposed and
flexible in the CD4-bound state, with an accordion-like stem, a
conserved fixed base, and a ?-hairpin tip (13). OD(9,9)(hCD4-
TM) has a V3 loop tip deletion and only retains 9 amino acids
on each base and the partial stem of V3 and is linked with
Gly-Val-Gly (15). To diminish the length of the stem and to
preserve the distance between residues on opposite sides of the
base, we deleted the V3 loop-stem closer to the base and
replaced it with one of several sequences, including a Gly-Ser
linker (GGSGSG [9c]), an acidic/negatively charged linker (E
NGEGE [AN]), a basic/positively charged linker (KNGKGK
[BP]), a glycan-shielded linker (GGNGSG [GS]), or a native
glycosylation site adjacent to a shorter/charged loop-stem (N
TRGRR [GSL]) (Fig. 3A and B). The OD-TM glycoproteins
with either Gly-Ser, acidic/negative, or glycan-shielded linkers
showed no increase in b12 and 2G12 binding compared to the
OD(9,9)(hCD4-TM) glycoprotein [9c, AN, or GS versus 9,9
OD(hCD4-TM)] (Fig. 3C). In contrast, the basic/positive or
glycosylation site adjacent to a shorter/charged loop linker
showed significantly increased binding to both b12 and 2G12,
with b12 recognition being even greater than 2G12 binding
(BP and GSL) (Fig. 3C). Thus, these V3 linkers improved the
relative exposure and reactivity of the CD4-BS determinant
recognized by b12.
Binding of CD4-BS antibodies to OD-TMs. An increasing
number of CD4-BS antibodies have been identified and char-
acterized, some of which neutralize diverse viruses and others
of which are weakly neutralizing or restricted in their breadth
(1, 3, 6, 12, 21, 24, 26, 27, 30–32, 37, 38). We tested
OD(GSL)(??20-21)(hCD4-TM), which showed increased
binding to b12. This OD-TM did not bind to CD4, CD4-
induced antibodies (17b), or most nonpotent neutralizing
CD4-BS antibodies, while these MAbs were able to bind to a
gp145 Env (b3, b6, b11, m14, m18, F91, F105, and 15e) (Fig. 4,
top versus bottom). The OD-TM did bind to neutralizing
CD4-BS antibody b12 as well as to one of the nonpotent
CD4-BS antibodies (b13) (Fig. 4, top). No binding, however, to
a D368R mutation that abolishes the interaction of CD4 with
Env was observed by flow cytometry (b12, b13, and wild type
versus D386R) (Fig. 4, middle).
We also tested the binding of OD(GSL)(??20-21)(hCD4-
TM) to five complex antisera (Fig. 5). One specimen (from
patient 1) was derived from a subject with broadly neutralizing
CD4-BS antibodies to HIV-1 (17). This antiserum along with a
second antiserum (from patient 20) failed to react with
OD(GSL)(??20-21)(hCD4-TM). Another antiserum (from
patient 45) showed marginal reactivity, whereas a fourth
(US21) showed moderate reactivity; however, with both of
these sera, similar reactivity to a D368R mutant was observed.
In contrast, antisera from another subject (Zeptometrix 1642)
with neutralizing activity specifically bound to this OD-TM but
not to the D368R mutant (Fig. 5). This specificity was not
apparent using a transmembrane form containing a nearly
complete Env that included the complete gp120 with a trun-
cated V3 region [gp145?CFI?V12(9,9); wild type versus
D368R] (Fig. 5), suggesting that the OD-TM eliminated irrel-
evant specificities and allowed the detection of specific
CD4-BS reactivity in complex antisera.
To determine whether these antibodies in Zeptometrix 1642
mediated HIV neutralization, we evaluated its neutralization ac-
tivity against HIV-1. Zeptometrix 1642 antisera showed neutral-
ization of HIV-1 strains from diverse clades (Table 1), and it thus
showed increased breadth compared to those of most sera (16).
This activity was nearly completely removed by OD-TM but not
the D368R mutant [OD(GSL)(??20-21)(hCD4-TM) versus
D368R] (Fig. 6A and B, left) when assayed against JRFL, a
CD4-BS antibody-sensitive virus. Finally, the CD4-BS reactivity
of OD-TM was confirmed with b12, whose neutralization activity
was removed by the wild type but not the D368R mutant (Fig. 6A
and B, right).
The development of immunogens that elicit broadly neutral-
izing antibodies remains a high priority for the development of
an effective AIDS vaccine. Among the targets of neutralization
is the highly conserved CD4-BS. MAb b12 binds primarily to
the surface of gp120 utilized in the initial contact with the CD4
receptor. This site is recessed on the viral spike, is surrounded
by both N-linked glycans and immunodominant variable loops,
and represents only one among many potential B-cell epitopes
One strategy for optimizing the presentation of this site is
“minimization,” to remove all immunogenic Env surfaces
other than the target site. Such minimization would have utility
both to define antibodies to this region in complex antisera and
to develop new prototypes as potential immunogens. However,
it has been a challenge to isolate the site of b12 binding inde-
pendent of the rest of the HIV Env, particularly the inner
domain. In this report, we show that an expression vector
encoding the OD and ?5-helix of Env linked to a transmem-
brane domain retains b12-binding activity and interacts specif-
ically with antisera with broadly neutralizing CD4-BS antibod-
ies. Absorption of one of those antisera selectively removed
neutralizing activity against several viruses. These data suggest
that it is possible to generate simplified proteins that can elim-
inate multiple specificities detected by anti-HIV-1 sera and to
focus recognition on the CD4-BS.
VOL. 83, 2009 TRANSMEMBRANE HIV gp120 OD BINDS b12 ANTIBODY 5081
FIG. 3. Differential charges at the base of V3 affect b12 recognition. (A) Structure of gp120 core with V3 and V3 sequences for the different
V3 loop mutations: original V3 from a R3A TA1 strain (9,9), V3 deletion with a Gly-Ser link (9c), V3 deletion with acidic/negative charge (AN),
V3 deletion with basic/positive charge (BP), V3 deletion with glycan shield (GS), and V3 deletion with native glycan and a shorter/charged
loop-stem (GSL). WT, wild type. (B) Models of V3 with the surface colored according to the chemistry of the underlying amino acids (red,
negatively charged; blue, positively charged; white, apolar). (C) MAb b12 (blue) or 2G12 (green) or human control IgG (red) binding to 293T cells
transfected with OD(hCD4-TM) with different V3 mutations was analyzed by flow cytometry.
5082WU ET AL.J. VIROL.
Previous studies have shown that a soluble outer domain
protein, OD1, is recognized by 2G12 and a number of V3
antibodies, but recognition by b12 was hampered by an en-
hanced off rate (36, 39). Laboratory-adapted virus strain R3A
TA1 was chosen for further modification because it was highly
sensitive to b12 neutralization and lacked most of the variable
loops. It is likely that similar results can be obtained by remov-
ing analogous Env regions in other strains, and while a soluble
OD based on R3A TA1 was similar to previously described
ODs (36; and data not shown), here, the addition of a cellular
transmembrane domain significantly improved b12 reactivity
as determined by flow cytometry. This feature was observed
with a variety of membrane anchors and was retained upon
modification of the V3 region. It is important to remove the V3
FIG. 4. Specific binding of ODs to CD4-BS MAbs. MAb (solid lines) or human control IgG (dashed lines) binding to 293T cells transfected
with OD(GSL)(??20-21)(hCD4-TM), its D368R mutation, and R3A gp145?CFI?V12(9,9) was analyzed by flow cytometry.
FIG. 5. Specificity of OD binding to selected neutralizing human antisera. Binding of MAb b12 (solid lines) and human sera or plasma from
HIV-positive patients (Pt.) (solid lines) to 293T cells transfected with OD(GSL)(??20-21)(hCD4-TM), gp145?CFI?V12(9,9), or their D368R
mutations was analyzed by flow cytometry. Human control IgG or normal human sera (dashed lines) were used as controls. SM, serum; PL, plasma.
VOL. 83, 2009TRANSMEMBRANE HIV gp120 OD BINDS b12 ANTIBODY5083
from candidate immunogens because of its strong but strain-
The OD-TM described here was generated after attempting
to develop secreted versions of OD. Secreted OD formed
aggregates when expressed in 293 cells and failed to interact
with IgG1 b12, similar to another soluble OD described pre-
viously (36). We reasoned that the loss of reactivity of soluble
OD was due in part to the loss of appropriate folding, a result
of inner domain removal. We further reasoned that an alter-
native surface might stabilize OD and that, possibly, a cell
membrane might provide a suitable surface. Thus, appropri-
ately anchoring OD to a membrane might stabilize it suffi-
ciently to retain a conformation recognized with high affinity by
b12. Other fusion protein approaches had failed to confer this
property, although it remains possible that secreted ODs could
be generated with heterologous domains using independent
The reasons for the improved reactivity of membrane-an-
chored OD are not fully known but include the possibility that
membrane anchoring allows protein contacts with the cell sur-
face that stabilize exposed regions that would otherwise inter-
act with the inner domain in the nontruncated Env (Fig. 2A to
E). In this context, the membrane may also occlude ligand
binding to the anchored OD in a manner similar to that of the
FIG. 6. Neutralization of HIVJRFLclade B virus by leukapheresis sera from an HIV-positive patient, Zeptometrix 1642, and absorption by
wild-type but not mutant OD-TM. (A) Zeptometrix 1642 antisera (left), b13 antibody (middle), and b12 antibody (right) were absorbed with 293F
cells transfected with OD(GSL)(??20-21)(hCD4-TM) (gray lines) or 293F cells transfected with OD(GSL)(??20-21)(hCD4-TM)(D368R) (solid
black lines). After absorption, residual binding was analyzed by flow cytometry for 293T cells expressing wild-type OD(GSL)(??20-21)(hCD4-TM).
Normal human serum (dashed lines) was used as a negative control. (B) Neutralizations of absorbed Zeptometrix 1642 antisera against HIV-1
clade B virus JRFL (left) and absorbed MAb b12 against HIV-1 clade B virus HxBc2 (right) were analyzed. Percent neutralization is shown for
antisera absorbed with 293F cells transfected with OD(GSL)(??20-21)(hCD4-TM) (diamonds) or with 293F cells transfected with
TABLE 2. Surface area calculations
Surface area (Å2)
Relative b12 surface
Total surfaceb12 epitope
aGlycan masking calculated with a 7-Å radius around each N-linked site of glycosylation (this radius results in full coverage of the gp120 silent face) (35).
bMembrane occlusion calculated by using a 4-Å radius of the inner domain in the core context.
5084WU ET AL.J. VIROL.
native viral spike. Finally, we note that cell surface avidity,
present in the membrane-anchored context but not the soluble
context, should reduce the off rate, thereby stabilizing b12
Interestingly, the V3 loop modification mutants tested here
showed that both the length and charge of V3 affect b12 bind-
ing. This result is somewhat unexpected since the V3 region
emanating from the core is distal from both the site of inner
domain truncation and the site of b12 recognition. To elimi-
nate the V3 immunodominance, we both truncated V3 and
made modifications to reduce its immunogenicity. In some
cases, modifications affected both 2G12 and b12 binding, indi-
cating an effect on OD-TM expression, but in others, b12
recognition was specifically enhanced, suggesting that its expo-
sure was increased relative to those of other epitopes on the
The deletion of the ?20-?21 hairpin has been suggested to
reduce steric hindrance around the CD4-BS, to expand the
CD4-binding pocket, and to increase b12 accessibility (2). Our
data showed that the deletion of the ?20-?21 hairpin improved
b12 and 2G12 binding. These data suggest that the ?20-?21
loop is not necessary for b12 binding to the membrane-an-
chored OD proteins, although the increased binding to 2G12
suggests that this deletion improves overall protein expression.
Both 2G12 and b12 are conformational antibodies (5, 29,
39). Our results showed that without the inner domain, the
outer domain of gp120 itself can form a stable structure that
allows those antibodies to become accessible. The optimized
ODs developed here specifically recognize b12 but not most
other nonpotent neutralizing CD4-BS antibodies. Moreover,
OD(GSL)(??20-21)(hCD4-TM) can absorb the HIV-1-neu-
tralizing antibody from the human sample Zeptometrix 1642.
Zeptometrix 1642 serum has reasonable breadth and potency,
although its activity is lower than that of patient 1 sera de-
scribed previously (17, 18). Other HIV-positive patient sera
have been well characterized, including sera from patients 1
and 45 (18). Both samples contain CD4-BS antibodies, but
their relationship to b12, b13, and Zeptometrix 1642 is un-
known, and they might be expected to differ since these sera
show broader reactivity across divergent clades than b12. A
possible explanation for the lack of binding of patient 1 serum
with the membrane-anchored OD is that it may require an
interaction with an additional region, such as the inner domain
or bridging sheet, not present in the outer domain. It is also
unclear whether the activity of patient 1 sera can be explained
by a single antibody or a combination of CD4-BS antibodies
directed against diverse epitopes. The identification of other
broadly neutralizing MAbs to the CD4-BS from such subjects
will be instructive in this regard.
Efforts to use OD-TM as a vaccine immunogen are currently
in progress. Structure-based calculations show that the propor-
tion of b12-reactive surface relative to the total exposed sur-
face on the optimized OD is significantly higher than that in
core gp120 or OD1 contexts (Table 2 and Fig. 7). Because of
its transmembrane nature, gene-based vaccination is required
to elicit immune responses. Nonetheless, the findings reported
here suggest that the membrane-anchored OD represents an
alternative form of the Env glycoprotein that can detect neu-
tralizing antibodies directed to the site of CD4 binding and
may contribute to the rational design of an AIDS vaccine.
This research was supported by the Intramural Research Program,
Vaccine Research Center, NIAID, NIH.
We thank Dimiter Dimitrov for m6, m14, and m18; Marshall Posner
for F105; James Robinson for 17b, F91, F105, and 15e; Ati Tislerics for
assistance with manuscript preparation; the NIH Fellows Editorial
Board for editorial assistance; Michael Cichanowski, Brenda Hartman,
and Jonathan Stuckey for assistance with figures; and members of the
Structural Biology Section and Virology Laboratory for helpful advice,
discussions, and comments on the manuscript.
1. Barbas, C. F., III, E. Bjorling, F. Chiodi, N. Dunlop, D. Cababa, T. M. Jones,
S. L. Zebedee, M. A. Persson, P. L. Nara, and E. Norrby. 1992. Recombinant
human Fab fragments neutralize human type 1 immunodeficiency virus in
vitro. Proc. Natl. Acad. Sci. USA 89:9339–9343.
2. Berkower, I., C. Patel, Y. Ni, K. Virnik, Z. Xiang, and A. Spadaccini. 2008.
Targeted deletion in the beta20-beta21 loop of HIV envelope glycoprotein
gp120 exposes the CD4 binding site for antibody binding. Virology 377:330–
3. Bublil, E. M., S. Yeger-Azuz, and J. M. Gershoni. 2006. Computational
prediction of the cross-reactive neutralizing epitope corresponding to the
monclonal [sic] antibody b12 specific for HIV-1 gp120. FASEB J. 20:1762–
4. Burton, D. R., R. C. Desrosiers, R. W. Doms, W. C. Koff, P. D. Kwong, J. P.
Moore, G. J. Nabel, J. Sodroski, I. A. Wilson, and R. T. Wyatt. 2004. HIV
vaccine design and the neutralizing antibody problem. Nat. Immunol. 5:233–
5. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren,
FIG. 7. b12 epitope relative surface area on core gp120, OD1, and
membrane-anchored OD. The b12 surface area was calculated in the
context of three immunogens: core gp120 (14), OD1 (36), and the
membrane-anchored OD developed here, OD(GSL)(??20-21)(hCD4-
TM). A model of the immunogen, with the b12 surface shown in blue,
and expected locations of glycan and membrane are shown. The rel-
ative surface area of the b12 epitope is shown, after immunogenic
masking by glycan and membrane components of the immunogens is
taken into account (Table 2). C-term, C terminus.
VOL. 83, 2009 TRANSMEMBRANE HIV gp120 OD BINDS b12 ANTIBODY5085
L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L. Nara, M. Lamacchia, E. Download full-text
Garratty, E. R. Stiehm, Y. J. Bryson, Y. Cao, J. P. Moore, D. D. Ho, and C. F.
Barbas. 1994. Efficient neutralization of primary isolates of HIV-1 by a
recombinant human monoclonal antibody. Science 266:1024–1027.
6. Cavacini, L. A., C. L. Emes, J. Power, A. Buchbinder, S. Zolla-Pazner, and
M. R. Posner. 1993. Human monoclonal antibodies to the V3 loop of HIV-1
gp120 mediate variable and distinct effects on binding and viral neutraliza-
tion by a human monoclonal antibody to the CD4 binding site. J. Acquir.
Immune Defic. Syndr. 6:353–358.
7. Chakrabarti, B. K., W. P. Kong, B.-Y. Wu, Z.-Y. Yang, J. Friborg, Jr., X.
Ling, S. R. King, D. C. Montefiori, and G. J. Nabel. 2002. Modifications of
the human immunodeficiency virus envelope glycoprotein enhance immu-
nogenicity for genetic immunization. J. Virol. 76:5357–5368.
8. 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.
9. Douek, D. C., P. D. Kwong, and G. J. Nabel. 2006. The rational design of an
AIDS vaccine. Cell 124:677–681.
10. Fauci, A. S., M. I. Johnston, C. W. Dieffenbach, D. R. Burton, S. M. Hammer,
J. A. Hoxie, M. Martin, J. Overbaugh, D. I. Watkins, A. Mahmoud, and
W. C. Greene. 2008. HIV vaccine research: the way forward. Science 321:
11. 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.
12. Ho, D. D., J. A. McKeating, X. L. Li, T. Moudgil, E. S. Daar, N. C. Sun, and
J. E. Robinson. 1991. Conformational epitope on gp120 important in CD4
binding and human immunodeficiency virus type 1 neutralization identified
by a human monoclonal antibody. J. Virol. 65:489–493.
13. Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L.
Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and
P. D. Kwong. 2005. Structure of a V3-containing HIV-1 gp120 core. Science
14. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A.
Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in
complex with the CD4 receptor and a neutralizing human antibody. Nature
15. Laakso, M. M., F. H. Lee, B. Haggarty, C. Agrawal, K. M. Nolan, M. Biscone,
J. Romano, A. P. Jordan, G. J. Leslie, E. G. Meissner, L. Su, J. A. Hoxie, and
R. W. Doms. 2007. V3 loop truncations in HIV-1 envelope impart resistance
to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies.
PLoS Pathog. 3:e117.
16. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos,
G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, M. Bilska, D. L. Kothe, J. F.
Salazar-Gonzalez, X. Wei, J. M. Decker, B. H. Hahn, and D. C. Montefiori.
2005. Human immunodeficiency virus type 1 env clones from acute and early
subtype B infections for standardized assessments of vaccine-elicited neu-
tralizing antibodies. J. Virol. 79:10108–10125.
17. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X.
Wu, G. M. Shaw, M. Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad
HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med.
18. Li, Y., K. Svehla, M. K. Louder, D. Wycuff, S. Phogat, M. Tang, S. A.
Migueles, X. Wu, A. Phogat, G. M. Shaw, M. Connors, J. Hoxie, J. R.
Mascola, and R. Wyatt. 2009. Analysis of neutralization specificities in poly-
clonal sera derived from human immunodeficiency virus type 1-infected
individuals. J. Virol. 83:1045–1059.
19. McRee, D. E. 1999. XtalView/Xfit—a versatile program for manipulating
atomic coordinates and electron density. J. Struct. Biol. 125:156–165.
20. Meissner, E. G., K. M. Duus, F. Gao, X. F. Yu, and L. Su. 2004. Character-
ization of a thymus-tropic HIV-1 isolate from a rapid progressor: role of the
envelope. Virology 328:74–88.
21. Moore, J. P., and J. Sodroski. 1996. Antibody cross-competition analysis of
the human immunodeficiency virus type 1 gp120 exterior envelope glyco-
protein. J. Virol. 70:1863–1872.
22. Nicholls, A., K. A. Sharp, and B. Honig. 1991. Protein folding and associa-
tion: insights from the interfacial and thermodynamic properties of hydro-
carbons. Proteins 11:281–296.
23. Nolan, K. M., A. P. Jordan, and J. A. Hoxie. 2008. Effects of partial deletions
within the human immunodeficiency virus type 1 V3 loop on coreceptor
tropism and sensitivity to entry inhibitors. J. Virol. 82:664–673.
24. Pantophlet, R., S. E. Ollmann, 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.
25. Pitisuttithum, P., P. Gilbert, M. Gurwith, W. Heyward, M. Martin, F. van
Griensven, D. Hu, J. W. Tappero, and K. Choopanya. 2006. Randomized,
double-blind, placebo-controlled efficacy trial of a bivalent recombinant gly-
coprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thai-
land. J. Infect. Dis. 194:1661–1671.
26. Posner, M. R., L. A. Cavacini, C. L. Emes, J. Power, and R. Byrn. 1993.
Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4
binding site of gp120. J. Acquir. Immune Defic. Syndr. 6:7–14.
27. Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R.
Burton. 1994. Recognition properties of a panel of human recombinant Fab
fragments to the CD4 binding site of gp120 that show differing abilities to
neutralize human immunodeficiency virus type 1. J. Virol. 68:4821–4828.
28. Russell, N. D., B. S. Graham, M. C. Keefer, M. J. McElrath, S. G. Self, K. J.
Weinhold, D. C. Montefiori, G. Ferrari, H. Horton, G. D. Tomaras, S.
Gurunathan, L. Baglyos, S. E. Frey, M. J. Mulligan, C. D. Harro, S. P.
Buchbinder, L. R. Baden, W. A. Blattner, B. A. Koblin, and L. Corey. 2007.
Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in
combination with rgp120: negative results fail to trigger a phase 3 correlates
trial. J. Acquir. Immune Defic. Syndr. 44:203–212.
29. Scanlan, C. N., R. Pantophlet, M. R. Wormald, S. E. Ollmann, R. Stanfield,
I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R. Burton. 2002.
The broadly neutralizing anti-human immunodeficiency virus type 1 anti-
body 2G12 recognizes a cluster of ?132 mannose residues on the outer face
of gp120. J. Virol. 76:7306–7321.
30. 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.
31. Thali, M., U. Olshevsky, C. Furman, D. Gabuzda, J. Li, and J. Sodroski.
1991. Effects of changes in gp120-CD4 binding affinity on human immuno-
deficiency virus type 1 envelope glycoprotein function and soluble CD4
sensitivity. J. Virol. 65:5007–5012.
32. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan,
K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human
monoclonal antibody 2G12 defines a distinctive neutralization epitope on the
gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70:
33. Walker, B. D., and D. R. Burton. 2008. Toward an AIDS vaccine. Science
34. Wu, L., Z. Y. Yang, L. Xu, B. Welcher, S. Winfrey, Y. Shao, J. R. Mascola,
and G. J. Nabel. 2006. Cross-clade recognition and neutralization by the V3
region from clade C human immunodeficiency virus-1 envelope. Vaccine
35. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A.
Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV
gp120 envelope glycoprotein. Nature 393:705–711.
36. Yang, X., V. Tomov, S. Kurteva, L. Wang, X. Ren, M. K. Gorny, S. Zolla-
Pazner, and J. Sodroski. 2004. Characterization of the outer domain of the
gp120 glycoprotein from human immunodeficiency virus type 1. J. Virol.
37. Zhang, M. Y., Y. Shu, S. Phogat, X. Xiao, F. Cham, P. Bouma, A. Choudhary,
Y. R. Feng, I. Sanz, S. Rybak, C. C. Broder, G. V. Quinnan, T. Evans, and
D. S. Dimitrov. 2003. Broadly cross-reactive HIV neutralizing human mono-
clonal antibody Fab selected by sequential antigen panning of a phage
display library. J. Immunol. Methods 283:17–25.
38. Zhang, M. Y., X. Xiao, I. A. Sidorov, V. Choudhry, F. Cham, P. F. Zhang, P.
Bouma, M. Zwick, A. Choudhary, D. C. Montefiori, C. C. Broder, D. R.
Burton, G. V. Quinnan, Jr., and D. S. Dimitrov. 2004. Identification and
characterization of a new cross-reactive human immunodeficiency virus type
1-neutralizing human monoclonal antibody. J. Virol. 78:9233–9242.
39. Zhou, T., L. Xu, B. Dey, A. J. Hessell, D. Van Ryk, S. H. Xiang, X. Yang,
M. Y. Zhang, M. B. Zwick, J. Arthos, D. R. Burton, D. S. Dimitrov, J.
Sodroski, R. Wyatt, G. J. Nabel, and P. D. Kwong. 2007. Structural definition
of a conserved neutralization epitope on HIV-1 gp120. Nature 445:732–737.
5086WU ET AL. J. VIROL.