HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120.
ABSTRACT HIV-1 envelope glycoprotein is the primary target for HIV-1-specific antibodies. The native HIV-1 envelope spike on the virion surface is a trimer, but trimeric gp140 and monomeric gp120 currently are believed to induce comparable immune responses. Indeed, most studies on the immunogenicity of HIV-1 envelope oligomers have revealed only marginal improvement over monomers. We report here that suitably prepared envelope trimers have nearly all the antigenic properties expected for native viral spikes. These stable, rigorously homogenous trimers have antigenic properties markedly different from those of monomeric gp120s derived from the same sequences, and they induce potent neutralizing antibody responses for a cross-clade set of tier 1 and tier 2 viruses with titers substantially higher than those elicited by the corresponding gp120 monomers. These results, which demonstrate that there are relevant immunologic differences between monomers and high-quality envelope trimers, have important implications for HIV-1 vaccine development.
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ABSTRACT: Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.Scientific reports. 01/2014; 4:7025.
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ABSTRACT: An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.PLoS ONE 01/2014; 9(10):e109196. · 3.53 Impact Factor
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ABSTRACT: Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.PLoS Pathogens 11/2014; 10(11). · 8.14 Impact Factor
HIV-1 envelope trimer elicits more potent neutralizing
antibody responses than monomeric gp120
James M. Kovacsa,b,1, Joseph P. Nkololac,d,1, Hanqin Penga, Ann Cheungc, James Perryc, Caroline A. Millerc,
Michael S. Seamanc, Dan H. Barouchc,d,2, and Bing Chena,b,2
aDivision of Molecular Medicine, Children’s Hospital, Boston, MA 02115;bDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115;cDivision of
Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02215; anddRagon Institute of MGH, MIT, and Harvard, Boston, MA 02114
Edited* by Michel C. Nussenzweig, The Rockefeller University, New York, NY, and approved June 13, 2012 (received for review March 16, 2012)
HIV-1 envelope glycoprotein is the primary target for HIV-1–spe-
cific antibodies. The native HIV-1 envelope spike on the virion
surface is a trimer, but trimeric gp140 and monomeric gp120
currently are believed to induce comparable immune responses.
Indeed, most studies on the immunogenicity of HIV-1 envelope
oligomers have revealed only marginal improvement over mono-
mers. We report here that suitably prepared envelope trimers
have nearly all the antigenic properties expected for native viral
spikes. These stable, rigorously homogenous trimers have anti-
genic properties markedly different from those of monomeric
gp120s derived from the same sequences, and they induce potent
neutralizing antibody responses for a cross-clade set of tier 1 and
tier 2 viruses with titers substantially higher than those elicited by
the corresponding gp120 monomers. These results, which demon-
strate that there are relevant immunologic differences between
monomers and high-quality envelope trimers, have important
implications for HIV-1 vaccine development.
fection (1). The precursor of the envelope protein, gp160, forms
a trimer and is then cleaved by a furin-like protease into two
noncovalently associated fragments: gp120 for receptor binding
and gp41 for membrane fusion. Three copies of each fragment
make up the mature envelope spike (gp120/gp41)3. This trimeric
complex undergoes large, irreversible structural rearrangements
after binding to the primary receptor CD4 and coreceptor (e.g.,
CCR5 and CXCR4) and drives the membrane fusion process.
Monomeric gp120 can dissociate from the complex either spon-
taneously or upon CD4 binding in certain viral strains (2).
The envelope glycoprotein also is the primary target of humoral
responses in HIV-1–infected patients. Strong evidence for the
potential of antibody protection comes from passive transfer and
mucosal simian-human immunodeficiency virus challenge studies
in macaques and from a vectored immunoprophylaxis study in
humanized mice (3–6). Although most antibodies induced during
infection are nonneutralizing or are strain specific, recent studies
indicate that 10–25% of patients produce broadly neutralizing
antibodies (bNAbs) during the course of HIV-1 infection (7),
raising the hope that immunogens capable of inducing such
responses may lead to an effective vaccine. A number of broadly
reactive neutralizing antibodies (NAbs) have been isolated that
recognize conserved regions of the envelope glycoprotein. mAbs
VRC01-03, CH31, 3BNC60, HJ16, and b12 target the CD4
binding site (CD4 BS) on gp120 with strong, broadly neutralizing
activity (reviewed in ref. 8); PG9 and PG16 appear to recognize
a quaternary epitope, which is trimer specific and glycan de-
pendent, in the relatively conserved regions of the variable V2
and V3 loops of gp120 (9); 2G12 is another bNAb that recog-
nizes an epitope on the outer surface of gp120 in a glycan- and
conformational-dependent manner (10). Very recently, another
group of bNAbs, designated “PGT antibodies,” has been identi-
fied; these antibodies react with glycan-dependent epitopes near
the base of the V3 loop (11). Additional bNAbs, including 2F5
and 4E10, interact with a region on gp41 adjacent to the viral
membrane called the “membrane-proximal external region”
(MPER) (12, 13). Among these bBAbs, those against gp120 are
IV-1 envelope glycoprotein mediates the fusion of viral and
target cell membranes, the first critical step leading to in-
believed to target directly the native, functional envelope trimer
on the surface of virion, whereas the gp41-directed antibodies
have been shown to block viral infection by attacking the pre-
hairpin intermediate conformation of gp41 (14, 15).
Anti-gp41 NAbs are rare both in natural infection and after
immunization with envelope-based vaccine candidates, and
gp120, in principle, contains most of the neutralizing epitopes.
Monomeric gp120 is relatively easy to manufacture and has been
used as a subunit vaccine in three large efficacy trials. In the two
early trials, gp120 vaccines failed to show any protection against
HIV-1 infection or delay in disease progression (16, 17). The
recent RV144 trial using a regimen involving priming with an
ALVAC vector and boosting with a gp120 protein afforded 31.2%
efficacy (18). A key question thus concerns the optimal form of the
envelope glycoprotein for inducing HIV-1–specific NAbs. Mo-
nomeric gp120 is safe and easy to manufacture, but there are
several reservations concerning its use as an immunogen. First,
gp120 vaccines alone provided little or no protection in human
efficacy trials (16–18). Second, antibodies elicited by monomeric
gp120 react mainly with epitopes that are poor neutralization
targets and presumably are occluded on primary HIV-1 isolates
(19). Third, the strongly immunogenic and ineffective epitopes
on monomeric gp120 could distract the immune system from
targeting the more relevant, broadly neutralizing epitopes.
Is the envelope trimer a better immunogen than the gp120
monomer? Cleaved and functional (gp120/gp41)3complexes are
unstable and are difficult to produce as recombinant products.
Thus, gp140, the ectodomain of trimeric gp160, has been used to
mimic the native state of the envelope spikes (20–23). Because
convincing evidence has been lacking to show that envelope
trimers or oligomers induce stronger NAb responses than mo-
nomeric gp120, there is a general belief that both forms of the
envelope glycoprotein have comparable immunogenicity. The
envelope trimers or oligomers used in previous immunogenicity
studies often had “gp120-like” characteristics, however, such as
binding to CD4-induced (CD4i) antibodies in the absence of
CD4 and exhibiting high affinity for nonneutralizing CD4 BS
antibodies (24–28). Moreover, they often either aggregate or
dissociate into dimers and monomers during expression or pu-
rification and probably fail to imitate the native envelope spikes.
We demonstrate here the feasibility of producing high-quality
HIV-1 envelope trimers in human cells with a yield suitable
for large-scale immunogenicity studies. We compare antigenic
properties of two biochemically stable and homogenous gp140
Author contributions: J.M.K., J.P.N., M.S.S., D.H.B., and B.C. designed research; J.M.K.,
J.P.N., H.P., A.C., J.P., and C.A.M. performed research; J.M.K., J.P.N., H.P., A.C., J.P.,
C.A.M., M.S.S., D.H.B., and B.C. analyzed data; and J.M.K., J.P.N., M.S.S., D.H.B., and B.C.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1J.M.K. and J.P.N. contributed equally to this work.
2To whom correspondence may be addressed. E-mail firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 24, 2012
| vol. 109
| no. 30
trimers with the corresponding gp120 monomers derived from
the same precursor sequences. We find that the gp140 trimers
react strongly only with anti-CD4 BS bNAbs but not with non-
neutralizing CD4 BS antibodies. We further show that the gp120
portions of the gp140 trimer maintain a conformation distinct
from the CD4-bound state and do not undergo conformational
changes upon binding to antibody VRC01. The trimers also show
detectable binding to the bNAbs PG9 and PG16, which fail to
react with the gp120 monomers. These stable trimers have dif-
ferent stoichiometries when binding soluble CD4 (1:1) and
VRC01 Fab (1:3). Moreover, the gp140 trimers induce potent,
cross-clade NAb responses with titers substantially higher than
those elicited by the corresponding gp120 monomers for a diverse
set of both tier 1 and tier 2 viruses. These results support the
concept that HIV-1 envelope trimers more accurately mimic the
antigenic properties of the native envelope spike on the surface of
virions than do gp120 monomers and thus are better immunogens.
Production of Stable gp140 Trimers and gp120 Monomers. We pre-
viously identified two sequences (92UG037.8 from clade A and
CZA97012 from clade C) for which the gp140 trimers are stable
and homogeneous when expressed in insect cells (6, 15). There
has been increasing recognition that N-linked glycans are im-
portant components of broadly neutralizing epitopes (9, 29, 30).
To produce these trimers with authentic human glycosylation, we
have generated stable 293T cell lines transfected with the ex-
pression constructs of 92UG037.8 and C97ZA012 gp140 trimers.
As shown in Fig. 1A, stable and homogenous gp140 proteins can
be purified from the supernatants of these cell lines, and the
yield for each of them is >5 mg/L. To compare antigenic and
immunogenic properties of the monomeric gp120 and trimeric
gp140 made from the same precursor sequences, we also
generated stable 293T cell lines that express gp120 from the
same isolates. The yield for the two monodisperse gp120
proteins is ∼8 mg/L (Fig. 1A). All purified proteins elute as
a single sharp peak from a sizing column and show a high level of
purity, as judged by SDS/PAGE stained by Coomassie (Fig. 1A,
Insets). Analytical ultracentrifugation and multiangle light-scat-
tering analyses confirm that gp140 is trimeric and gp120 is mo-
nomeric (Table S1). The gp140 trimers do not dissociate into
dimers or monomers during or after purification, and they re-
main stable at 4 °C for at least 4 wk. As expected, all the gp140
trimers and gp120 monomers bind tightly to soluble CD4, as
detected by surface plasmon resonance (SPR) binding experi-
ments (Fig. 1B, Fig. S1, and Table S2).
Antigenic Properties of Trimeric gp140 and Monomeric gp120. CD4 BS
epitopes.The CD4 BS is one of the most vulnerable regions of the
envelope protein. It is targeted by a number of bNAbs. On the
native, functional envelope trimer, the site probably is accessible
only to CD4 and bNAbs and inaccessible to nonneutralizing
mAbs, but on monomeric gp120 it is well exposed and open to
all CD4 BS ligands (31). We used SPR to assess reactivity of
VRC01, one of the most potent bNAbs targeting the CD4 BS, to
both gp140 and gp120. To avoid potential artifacts associated
with chemical immobilization, we used the human antibody
binder, a mixture of monoclonal antibodies recognizing the Fab
region of human antibodies, to capture and orient the VRC01
Fab fragments. As shown in Fig. 2A and Fig. S2, all the gp140
trimers and gp120 monomers bind tightly to the VRC01 Fab,
with affinities ranging from 2.1 to 96 nM. The slower off-rate,
and hence higher affinity, for the VRC01–gp140 interactions,
compared with those of VRC01–gp120s interactions, may result
from avidity effects when the monomeric Fab is immobilized on
the sensor chip. Both the gp140 and gp120 proteins derived from
the clade A isolate also react strongly with another CD4 BS bNAb,
CH31, but the proteins from the clade C isolate bind CH31 less
tightly, consistent with the lower sensitivity of this isolate to
CH31 neutralization (Fig. S3 and Table S3). In contrast, a non-
neutralizing CD4 BS antibody, b6, reacts with the 92UG037.8
gp120 with an affinity of 50 nM but does not bind at all to the
92UG037.8 gp140 trimer (Fig. 2A), indicating that the b6 epitope
is effectively occluded by the trimer configuration. Thus, the
conformation of the CD4 BS in our stable gp140 trimers is
consistent with the neutralization profile of a native, functional
CD4i epitopes. CD4 binding induces the formation of the bridging
sheet in gp120, creating the coreceptor binding site as well as
CD4i epitopes (32–34). CD4i antibodies, such as 17b, often are
used to detect conformational changes in gp120. In Fig. 2B,
immobilized 17b Fab can capture the full-length gp120 even in
the absence of CD4, suggesting that the free monomeric gp120 is
not fully rigid and that it samples the CD4-bound conformation
in solution. As expected, CD4 binding greatly enhances the
strength of the interaction between gp120 and 17b (Fig. 2B, and
Fig. S4, and Table S2). In contrast, the gp140 trimer does not
bind the 17b Fab at all in the absence of CD4, even at high Fab
concentrations, but binds tightly in the presence of soluble CD4
(Fig. 2B, Fig. S4, and Table S2). Thus, the gp120 portions in the
unliganded envelope trimer are tightly confined in a conformation
distinct from the CD4-bound state.
VRC01 induces conformational changes in gp120 similar to those
triggered by CD4, but it does not induce those changes in the
functional envelope trimers expressed on the cell surfaces (35).
We asked whether VRC01 can induce conformational changes
in our recombinant gp120 and gp140. As shown in Fig. 2B and
Fig. S4, 17b indeed binds the preformed gp120-VRC01 Fab
complex more tightly than does gp120 alone, but it does not
bind the gp140-VRC01 Fab complex at all (also see Table S2).
CD4 / 92UG037.8 gp120
CD4 / 92UG037.8 gp140
in 293T cells. (A) His-tagged gp140 proteins derived from the HIV-1 92UG037.8
(clade A) and CZA97012 (clade C) isolates and their corresponding full-length
gp120s were purified from supernatants of 293T cells stably transfected with
the expression constructs of these proteins. The purified gp120(Left) and gp140
(Right) proteins were resolved by gel-filtration chromatography on a Superdex
200 and a Superose 6 column, respectively. The molecular weight standards
include thyoglobulin (670 kDa), ferritin (440 kDa), γ-globulin (158 kDa), and
ovalbumin (44 kDa). Peak fractions were pooled and analyzed by Coomassie-
stained SDS/PAGE (Insets). (B) Soluble 4D CD4 was immobilizedon a CM-5chip,
and various concentrations (50–1,000 nM) of 92UG037.8 gp120 or 92UG037.8
gp140 were passed over the chip surface. Binding kinetics was evaluated using
a 1:1 Langmuir binding model; binding constants are summarized in Table S2.
The sensorgrams are shown in black and the fits in green. All injections were
carried out in duplicate and gave essentially identical results. Only one of the
duplicates is shown.
Production of stable and homogenous HIV-1 gp120 and gp140 proteins
| www.pnas.org/cgi/doi/10.1073/pnas.1204533109Kovacs et al.
These results also are confirmed by ELISA. We conclude that
our stable gp140 trimers, like the cell-surface–expressed en-
velope trimers, resist conformational changes induced by
VRC01 binding (35).
Quaternary epitopes. The intact quaternary epitope, first defined by
antibodies 2909, PG9, and PG16, is believed to form only on
the surface of the functional trimer (9, 36). Binding of PG9/16
to soluble envelope monomers as well as to trimers has been re-
ported, and a crystal structure of PG9 in complex with an isolated
V1V2 loop presented on an unrelated scaffold also has been
determined (9, 37–39). However, the reported PG9/16 binding is
not strictly trimer specific, nor does the binding fully correlate
with neutralization. In our experiments, both the 92UG037.8
and C97ZA012 gp140 trimers show detectable binding to PG9 and
PG16 (Fig. 3A), whereas monomeric gp120s derived from the two
isolates fail to bind either of these antibodies. Thus, the trimer
organization of all gp140 species restores, at least partially, the
epitopes targeted by PG9 and PG16. Interactions of the PG 9/16
antibodies to our trimers are still weaker than their neutrali-
zation potency (Table S3 and Fig. 3B) but are comparable to
the affinity of these antibodies for the monomeric A244 gp120
gD(+), used in the RV144 trial, which has been reported to
bind these antibodies tightly (38). Moreover, the binding pat-
tern of the trimers to PG9 and P16 agrees with the neutralization
profile of the two antibodies for these isolates (Table S3). For
example, the 92UG037.8 gp140 trimer binds more tightly to PG9
and PG16 than does the C97ZA012 gp140, consistent with the
greater sensitivity of the 92UG037.8 isolate to neutralization by
these antibodies. Antibody binding of our gp140 trimers thus
correlates directly with neutralization of the purified antibodies.
As shown in Fig. 3B, the affinities of the two gp140 trimers for
the two antibodies range from 43 to 310 nM.
Glycan-dependent neutralizing epitopes. Additional broadly neutral-
izing epitopes on gp120 require N-linked glycans, in particular, at
the residue Asn332 on the outer domain of gp120, which is
covered by densely packed carbohydrates. These antibodies ei-
ther must bind directly to the carbohydrates or penetrate the
glycan shield (30). As shown in Fig. S5, 2G12 binds gp120 and
gp140 almost equally well, although gp140 shows a slower off-
rate, perhaps because of avidity effects when the Fab is immo-
bilized. Likewise, another bNAb, PGT123, targeting the V3 loop
stem, also reacts well with both gp120 and gp140 (Figs. S5 and
S6), indicating that these epitopes are fully accessible in both
monomeric and trimeric forms of the envelope protein.
Finally, as expected, the mammalian cell–expressed 92UG037.8
trimer does not bind MPER-directed bNAbs, which target the
fusion intermediate state of gp41 (Fig. S7), and binds only weakly
to anti-gp41 cluster II antibodies (40). Overall, our stable and
homogeneous gp140 trimers exhibit a number of characteristics
expected for a native, functional envelope trimer.
Stoichiometry of the interactions of gp140 trimers with the CD4 BS
ligands. To characterize further the binding of the gp140 trimers
with the CD4 BS ligands, we determined the stoichiometry of
these interactions by analytical ultracentrifugation and multi-
angle light scattering. Values of the molecular mass of the same
protein or protein complex determined by the two independent
approaches generally were in excellent agreement with each
other (Table S1 and Figs. S8 and S9). The molecular masses
determined for the two gp140s were ∼412 kDa, as expected for
VRC01 Fab / 92UG037.8 gp140
VRC01 Fab / 92UG037.8 gp120
b6 Fab / 92UG037.8 gp120b6 Fab / 92UG037.8 gp140
17b Fab / 92UG037.8 gp120
17b Fab / (92UG037.8 gp120
17b Fab / (92UG037.8 gp120
+ VRC01 Fab)
17b Fab / (92UG037.8 gp140
+ VRC01 Fab)
17b Fab / (92UG037.8 gp140
17b Fab / 92UG037.8 gp140
antibody. (A) Fab of the CD4 BS antibody VRC01 or b6 was captured on
a surface coated with human Fab binder, and varous concentratons(50–1,000
nM) of 92UG037.8 gp120 or 92UG037.8 gp140 were passed over the chip
surface. (B) Fab of the CD4i antibody 17b was immobilized on a chip surface
using human Fab binder. Then 92UG037.8 gp120 and 92UG037.8 gp140, pu-
rified 92UG037.8 gp120-CD4 and 92UG037.8 gp140-CD4 complexes, or puri-
fied 92UG037.8 gp120-VRC01 Fab and 92UG037.8 gp140-VRC01 Fab complexes
at various concentrations (50–1,000 nM) were passed over the 17b surface.
Binding kinetics was evaluated using a 1:1 Langmuir binding model; binding
constants are summarized in Table S2. The sensorgrams are shown in black and
the fits in green. RU, response units.
Interactions of gp140 and gp120 with CD4 BS antibodies and CD4i
PG16 IgG / Envelope
A244 gp120 gD (+)
PG9 IgG / Envelope
A244 gp120 gD (+)
PG16 IgG / 92UG037.8 gp140
PG16 IgG / C97ZA012 gp140
PG9 IgG / 92UG037.8 gp140
PG9 IgG / C97ZA012 gp140
epitopes. (A) The bNAb PG9 IgG (Left) or PG16 IgG (Right) was captured on
a chip surface coated with protein A. Sensorgrams were recorded by passing
92UG037.8 gp140 trimer (1 μM; in red); C97ZA012 gp140 trimer (1 μM; in
blue); 92UG037.8 gp120 monomer (1 μM; in dark red); C97ZA012 gp120
monomer (1 μM; in dark blue); or A244 gp120 gD(+) monomer (1 μM; in
black) over the antibody surfaces. The apparent differences in response units
are caused partly by differences in molecular masses of the flowing analytes.
(B) PG9 (Left) or PG16 IgG (Right) was immobilized on the chip surface using
human Fab binder. Then various concentrations (50–1,000 nM) of 92UG037.8
gp140 or C97ZA012 gp140 were passed over the antibody surfaces. Binding
kinetics was evaluated using a 1:1 Langmuir binding model; binding con-
stants are summarized in Table S2. The sensorgrams are shown in black and
the fits in green.
Binding of gp140 and gp120 to bNAbs against the quaternary
Kovacs et al.PNAS
| July 24, 2012
| vol. 109
| no. 30
a trimer, and were ∼127 kDa for the two monomeric gp120s. The
molecular masses determined for the complexes of four-domain
CD4 with gp140 trimers were ∼456 kDa, showing there is only
one bound CD4 molecule per trimer in the complex. In contrast,
the mass determined for the VRC01 Fab-gp140 complex was
∼567 kDa, corresponding to a stoichiometry of 3:1 for the
VRC01–gp140 interaction. In addition, the measured molecular
mass of the gp140-CD4-17b complexes was ∼508 kDa. Thus, it
appears that one CD4-triggered gp140 trimer can bind just one
17b Fab, suggesting that the other two gp120 subunits preserve
their unliganded conformations.
Immunogenicity in Guinea Pigs. Binding antibody responses. To eval-
uate the immunogenicity of these gp120s and gp140s, we immu-
nized outbred female Hartley guinea pigs (n = 5 per group) at
weeks 0, 4, 8, 20, 24, and 28 by i.m. injection with 100 μg protein
per animal, using immunostimulatory di-nucleotide CpG DNA
and 15% (vol/vol) oil-in-water emulsion Emulsigen/PBS as ad-
juvant. This adjuvant combination has been shown to give a more
balanced immune response with higher levels of NAbs than each
adjuvant on its own (41). Because the molar ratio of gp120 in the
monomer immunogen and the gp120 moiety in the trimer im-
munogen was 1.2:1, the effective dosage of gp120 was slightly
higher in the monomer than in the trimer. Envelope-specific
antibodies to all four immunogens were assessed by ELISA of sera
obtained 4 wk after each immunization. High-titer binding anti-
body responses were observed in all immunized animals, but
responses after a single immunization were higher in the animals
that received the gp140 trimers (Fig. 4A and Fig. S10A). Binding
antibody titers induced by the monomer and trimer were com-
parable after the third immunization.
NAb responses. We compared neutralizing antibody responses eli-
cited by our stable gp140 trimers and corresponding gp120 mon-
omers using a standardized multiclade panel of tier 1 pseudoviruses
from clades A, B, and C in the TZM.bl assay (42) as well as tier 2
clade C infectious molecular clone (IMC) viruses in the A3R5
assay. NAb responses were assessed at baseline and after three and
six immunizations. We defined the criteria for NAb positivity as
ID50titers that were (i) more than threefold above preimmune
background, (ii) more than twofold above a concurrent murine
leukemia virus (MuLV) control, and (iii) an absolute titer >60.
Guinea pigs immunized with either the 92UG037.8 or
C97ZA012 gp140 trimer developed robust, cross-clade neutral-
izing activity against several tier 1 clade A (DJ263.8), clade B
(SF162.LS and BaL.26), and clade C (MW965.26, TV1.21,
ZM109F.PB4, and ZM197M.PB7) viruses with ID50 titers
against the most sensitive virus in the panel, MW965.26, ranging
from 57,247–196,648 after the third immunization and from
113,956–848,672 after the sixth immunization (Fig. 4B and Figs.
S10B and S11). Tier 1 NAb titers also were elicited by gp120
monomers, but the overall magnitudes of these titers were sub-
stantially lower than those observed for their trimeric counter-
parts (Fig. 4B and Fig. S10B). Specifically, mean ID50 titers
induced by the C97ZA012 gp140 trimer were 3.3-, 4.1-, 15.1-,
and 3.4-fold greater than median titers induced by the gp120
monomer after the third immunization against MW965.26,
DJ263.8, SF162.LS, and BaL.26, respectively, and were 15.1-,
9.9-, 9.0-, and 3.4-fold greater than mean titers induced by the
gp120 monomer after the sixth immunization against these same
viruses (Fig. 4B). Statistical analyses confirmed that C97ZA012
trimers gave significantly higher tier 1 NAb titers than did their
monomeric counterparts [P < 0.01 (DJ263.8, SF162.LS, BaL.26,
TV1.21, and ZM109F.PB4) and P < 0.05 (MW965.26)]. Similar
results were observed with the 92UG037.8 gp140 trimer [P <
0.01 (DJ263.8, SF162.LS, MW965.26, TV1.21, and ZM109F.
PB4)] (Fig. S10B).
We could not detect NAb activity in the TZM.bl assay against
two neutralization-resistant tier 2 clade C Env pseudoviruses
(ZM233M.PB6 and Du422.1) with sera from animals immunized
with either gp120 monomer or gp140 trimer (Fig. S11). We then
tested sera against three tier 2 clade C IMC viruses using the
more sensitive A3R5 neutralization assay (Fig. 4C and Figs.
S10C and S12). We observed that the C97ZA012 gp140 trimer
induced higher frequencies of NAb responses as well as higher
ID50titers than did the gp120 monomer against the tier 2 viruses
Du422.1 (P < 0.05), Ce1086_B2 (P = NS), and Ce2010_F5 (P <
0.05) (Fig. 4C). As expected, tier 2 NAb titers were lower than
tier 1 NAb titers but were clearly detectable in this assay. Taken
together, these data demonstrate that in guinea pigs the gp140
trimers elicited higher-magnitude tier 1 and tier 2 NAbs as well
as more rapid kinetics of antibody responses than did the cor-
responding monomeric gp120s.
The recent RV144 trial has provided evidence that an HIV-1
vaccine containing an envelope protein boost might afford
some degree of protection against HIV-1 infection in clinical
C97ZA012 Env: Pre-Bleed (TZM.bl assay)
C97ZA012 Env: Post-3x Immunization (TZM.bl assay)
C97ZA012 Env: Post-6x Immunization (TZM.bl assay)
ID50 Titer (1/x)
ID50 Titer (1/x)
ID50 Titer (1/x)
C97ZA012 Env (A3R5 assay)
gp120 Monomer gp140 Trimer
ID50 Titer (1/x)
Pre 1 2 3 4 5 6
Pre 1 2 3 4 5 6
gp120 in guinea pigs. (A) Sera of guinea pigs, vaccinated with either
C97ZA012 gp120 or gp140 obtained prevaccination (Pre) and 4 wk after each
immunization, were tested in endpoint ELISAs against all 92UG037.8 and
C97ZA012 antigens as indicated. Binding antibody responses against
92UG037.8 gp120 monomer (white bars), 92UG037.8 gp140 trimer (black
bars), C97ZA012 gp120 monomer (light gray bars), and C97ZA012 gp140
trimer (dark gray bars) are presented as geometric mean titers at each time
point ± SD. Horizontal lines indicate background threshold. (B) Guinea pig
sera obtained prevaccination (Pre-Bleed), 4 wk after the third vaccination
(Post-3×), and 4 wk after the sixth vaccination (Post-6×) were tested against
a multiclade panel of tier 1 neutralization-sensitive isolates, including clade
A (DJ263.8), clade B (SF162.LS and Bal.26), and cladeC (MW965.26, TV1.21,
ZM109F, and ZM197M) HIV-1 Env pseudoviruses and MuLV (negative con-
trol) in TZM.bl neutralization assays. NAb titers induced by C97ZA012 gp120
and C97ZA012 gp140 are represented graphically. *P < 0.05; **P < 0.01;
unpaired two-tailed t test. Horizontal bars indicate median titers. (C) Sera
obtained prevaccination (Pre) and 4 wk after the sixth vaccination (Post-6×)
were tested against three tier 2 clade C isolates (Du422, Ce1086_B2, and
Ce2010_F5 IMC viruses) in A3R5 neutralization assays. NAb titers induced by
C97ZA012 gp120 and C97ZA012 gp140 are represented y graphically. *P <
0.05; unpaired two-tailed t test.
Immunogenicity of the C97ZA012 trimeric gp140 and monomeric
| www.pnas.org/cgi/doi/10.1073/pnas.1204533109Kovacs et al.
settings (18). It remains uncertain, however, which form of the
envelope glycoprotein is better at inducing NAbs. Our current
immunogenicity studies in guinea pigs clearly show that the
gp140 trimers induce potent, cross-clade NAb responses with
titers that are substantially higher than those induced by the
gp120 monomers for a diverse set of both tier 1 and tier 2
viruses. We conclude that the HIV-1 envelope trimer is indeed
a better immunogen than the monomer and suggest that the
well-characterized gp140 trimers should be considered for fu-
ture clinical trials.
Env-based immunogens, including gp120 and gp140, have
been tested extensively in animal models and induce high titers of
binding antibodies, typically with limited potency and breadth of
neutralizing activity (19). Several studies comparing the immu-
nogenicity of monomeric gp120 and trimeric or oligomeric gp140
showed only marginal improvement when the latter was used
(24–27, 43), leading to the widely held belief that these immu-
nogens are comparable. Most gp140s in the previous immuno-
genicity studies had suboptimal stability (26, 44, 45) and also
exhibited gp120-like characteristics, such as binding to CD4i anti-
bodies in the absence of CD4 and interacting with nonneutralizing
CD4 BS antibodies. Some also reacted with MPER-directed NAbs
(46). Binding CD4i antibodies in the absence of CD4 or interacting
with nonneutralizing CD4 BS antibodies is not expected for the
native envelope spikes, because such binding will compete directly
with receptor or coreceptor and confer high neutralization po-
tency. Sophisticated molecular design was required to produce
a “resurfaced” gp120 protein or a gp140 variant that can discrim-
inate the neutralizing and nonneutralizing CD4 BS antibodies
(28, 47), but our stable trimers distinguish between these two types
of antibodies (Fig. 2). The MPER-directed antibodies neutralize
by targeting the prehairpin intermediate conformation of gp41,
and their epitopes are not present on the native envelope spikes
(14, 15). Thus, reactivity with MPER antibodies by a gp140 prep-
aration, which supposedly mimics the untriggered state of the
envelope, would indicate that parts of the gp41 moiety in the
prepared protein are very flexible or that they have undergone
unintended conformational changes. Some gp140 trimers re-
ported previously probably contain three gp120s with few mutual
structural constraints, held together only by a common gp41
“stem.” Therefore it is not surprising that these gp140 proteins
show gp120-like characteristics and exhibit immunogenicity
similar to that of the corresponding gp120 monomers. From the
data presented here, we suggest that a gp140 trimer in the pre-
fusion conformation should have the following characteristics: (i)
high stability and conformational homogeneity; (ii) no binding
of CD4i antibodies in the absence of CD4; (iii) discrimination
between neutralizing and nonneutralizing CD4 BS antibodies; (iv)
no conformational changes upon binding to VRC01; (v) re-
activity with bNAbs, such as PG9 and PG16, that target the
trimer-dependent, quaternary epitopes; and (vi) no interaction
with MPER-directed antibodies. Of course there may be other
distinct antigenic properties associated with the fully cleaved,
functional envelope trimer.
The stoichiometry (3:1) of VRC01 binding to the stable gp140
trimers confirms that the three CD4 BS in the trimer are well
exposed and that they can accommodate three molecules with
the size of an Fab. Why, then, can only one CD4 molecule bind
to an envelope trimer? Reinherz and colleagues first reported
the stoichiometry of CD4 binding to a stable SIV gp140 trimer
as 1:1 (48) but later concluded that one HIV-1 ADA strain gp140
trimer can bind more than one CD4 molecule (49). Those in-
vestigators postulated that the HIV-1 envelope may have evolved
to be more open, closer to a “fusion-ready” transition than the
simian immunodeficiency virus (SIV) envelope, implying that
HIV-1 envelope may be more sensitive to antibody neutraliza-
tion. We now have demonstrated that the stable HIV-1 gp140
trimers have the same stoichiometry for CD4 binding as the SIV
trimer, suggesting that this phenomenon may be physiologically
relevant. VRC01 binding, with a stoichiometry of 3:1, does not
induce conformational changes in gp140 trimers, whereas CD4,
which reacts with only one gp120 and breaks the symmetry of the
trimer, triggers large structural rearrangements in gp120. This
observation indicates that the asymmetry introduced by CD4 is
caused by the conformational changes induced by CD4 binding,
not by steric interference. Crystallographic analysis of gp120 core
proteins revealed that CD4 binding induces formation of the
bridging sheet as well as reorganization of the inner domain of
gp120 and possibly large movements of the V1V2 loop (34).
Conceivably, these movements can block the other two CD4 BS,
thereby creating an asymmetric structure. Moreover, the asym-
metry of CD4-liganded gp140 prevents the other subunits from
undergoing similar structural rearrangements, because only one
17b Fab binds a CD4-triggered trimer (Table S1). Srivastava
et al. (45) reported that all three CD4 BS are available to CD4
on certain gp140 trimers that have a deletion in the V2 loop.
Thus, modifications in the variable loop can change the valency
of the gp140 trimers for CD4 association.
If our observations on the CD4-induced asymmetry are indeed
relevant to HIV-1 entry, what are the advantages of such nega-
tive cooperativity of CD4 binding for the functional trimer?
First, requiring only one CD4 and, presumably, one coreceptor
to trigger the envelope trimer is an efficient way to initiate
membrane fusion, given the scarcity of the envelope spikes on
the surface of virion (50). Second, blocking the other two CD4
BS and preventing them from further conformational changes
would effectively reduce the number of vulnerable sites during
the fusion-intermediate steps that can be attacked by the host
immune responses. Thus, the unusual stoichiometry by the re-
ceptor binding may be still another mechanism by which HIV-1
evades the host immune system. Third, three CD4-induced
rearrangements might destabilize the trimer and lead to pre-
mature gp120 shedding, thereby inactivating it.
The bNAbs isolated from infected patients show remarkable
potency and breadth. Induction of such responses is a critical goal
for vaccine development, but none of the envelope-based
immunogens, including our stable gp140 trimers presented here,
has been able to raise high levels of bNAbs in small animal
models. Detailed analysis of these monoclonal antibodies suggests
that somatic hypermutation, as well as breaking host tolerance to
carbohydrates or lipids, may be required for developing potent
broadly neutralizing activities over an extended period (9, 11, 14,
51). We note that the broadly neutralizing epitopes involving N-
linked glycans on the rigid surface of the gp120 outer domain
appear to be well exposed on both monomeric gp120 and tri-
meric gp140 but failed to induce high titers of 2G12- or PGT123-
like antibodies in the current study. Moreover, significant pro-
tection against stringent virus challenges can be achieved in
nonhuman primates even without high titers of NAbs that
neutralize resistant isolates (52). Thus, the full potential of our
trimer immunogens may need to be evaluated further in more
relevant clinical settings. Overall, we believe that generating
improved trimer immunogens that closely mimic the native
functional spikes on the HIV-1 virion is an important step toward
an effective HIV-1 vaccine.
Methods and Materials
Production of HIV-1 Envelope Proteins, CD4, and Antibodies. Expression con-
structs and protein production of HIV-1 envelope protein, CD4, and antibodies
are described in SI Materials and Methods. All envelope proteins used were
produced in 293T cells.
SPRBinding Assays, Multiangle Light Scattering, and Analytical Ultracentrifugation.
Details of all binding experiments, multiangle light scattering, and analytical
ultracentrifugation are described in SI Materials and Methods.
Animals, Immunizations and ELISA. Details of animals, immunizations, and
ELISA are described in SI Materials and Methods.
NAb Assays. NAb responses against HIV-1 Env pseudoviruses were measured
using luciferase-based neutralization assays in TZM.bl cells as described (6, 42).
Details are given in SI Materials and Methods. For neutralization assays in
Kovacs et al.PNAS
| July 24, 2012
| vol. 109
| no. 30
A3R5 cells, serial dilutions of serum samples were performed in 10% RPMI
growth medium (100 μL per well) in 96-well flat-bottomed plates. IMC HIV-1
expressing Renilla luciferase (53) was added to each well in a volume of 50 μL,
and plates were incubated for 1 h at 37 °C. Then A3R5 cells were added (9 ×
104cells per well in a volume of 100 μL) in 10% RPMI growth medium con-
taining diethylaminoethyl-dextran (11 μg/mL). Assay controls included repli-
cate wells of A3R5 cells alone (cell control) and A3R5 cells with virus (virus
control). After incubation for 4 d at 37 °C, 90 μL of medium was removed from
each assay well, and 75 μL of cell suspension was transferred to a 96-well
white, solid plate. Diluted ViviRen Renilla luciferase substrate (Promega) was
added to each well (30 μL), and after 4 min the plates were read on a Victor 3
luminometer. The A3R5 cell line was generously provided by R. McLinden and
J. Kim (US Military HIV Research Program, Rockville, MD). Tier 2 clade C IMC
Renilla luciferase viruses Du422.1.LucR.T2A.ecto, Ce2010_F5.LucR.T2A.ecto,
and Ce1086_B2.LucR.T2A.ecto were generously provided by C. Ochsenbauer
(University of Alabama at Birmingham, Birmingham, AL), and stocks were
prepared in 293T/17 cells as previously described (53).
ACKNOWLEDGMENTS. We thank Stephen Harrison, Michael Freeman,
Sophia Rits-Volloch, Joanna Mangar, and Gary Frey for generous advice
and assistance; and Larry Liao, Barton Haynes, Dennis Burton, John Mascola,
and James Robinson for reagents. This work was supported by National
Institutes of Health Grants AI084794, AI078526, and GM083680; the Ragon
Institute of MGH, MIT, and Harvard; and the Center for HIV/AIDS Vaccine
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| www.pnas.org/cgi/doi/10.1073/pnas.1204533109Kovacs et al.