JOURNAL OF VIROLOGY, Feb. 2006, p. 1680–1687
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 4
Structure-Function Analysis of the Epitope for 4E10, a Broadly
Neutralizing Human Immunodeficiency Virus Type 1 Antibody†
Florence M. Brunel,1Michael B. Zwick,2Rosa M. F. Cardoso,3Josh D. Nelson,2
Ian A. Wilson,3,4Dennis R. Burton,2,3and Philip E. Dawson1,4*
Departments of Chemistry and Cell Biology,1Department of Immunology,2Department of Molecular Biology,3
and Skaggs Institute for Chemical Biology,4The Scripps Research Institute, La Jolla, California 92037
Received 18 May 2005/Accepted 27 November 2005
The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly
conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We
have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using
peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide bind-
ing to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the Kdvalues
of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated
that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement
with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make
significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported
crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses.
Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH2could effectively
inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting
2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have signifi-
cantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more
likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative
development of a vaccine based on the 4E10 epitope.
A major goal in human immunodeficiency virus type 1
(HIV-1) vaccine development is to elicit broadly neutralizing
antibodies (6, 20, 25). Such antibodies target conserved
epitopes on the HIV-1 surface glycoprotein gp120 and the
transmembrane glycoprotein gp41, which interact nonco-
valently to form a trimer of heterodimers on the virion surface
(10, 37). A few broadly neutralizing human monoclonal anti-
bodies (MAbs) against gp120 (6a, 35a) and against gp41 have
been identified (34, 42). In particular, MAbs 2F5, Z13, and
4E10 recognize conserved linear epitopes in the membrane-
proximal external region of gp41, and these epitopes have been
identified as promising vaccine leads (39).
However, design of immunogens able to elicit antibodies
akin to the anti-gp41 neutralizing MAbs has proven elusive.
For instance, antibodies elicited against recombinant synthetic
gp41 and sequences corresponding to the 2F5 core linear
epitope are typically nonneutralizing (12, 15, 19, 22, 24, 27).
This lack of success may be a result of the failure of the
synthetic gp41 peptides to adopt a conformation similar to that
of the corresponding peptide epitopes on gp41 prior to, or
during, the fusion process. Restricting the peptide to adopt a
specific ensemble of relevant conformations might enhance the
probability of eliciting neutralizing antibodies. The epitope for
the neutralizing antibody 2F5 adopts a largely extended pep-
tide structure, and mimicking such a conformation is quite
challenging (29). In contrast, a recent crystallographic struc-
ture suggests that the 4E10 epitope adopts a largely helical
structure, which is more amenable to structural constraint (9).
Antibody 4E10 is the most broadly neutralizing monoclonal
antibody that has been discovered, as characterized by a sensitive,
single-round infectivity assay (3, 26). Thus, the 4E10 epitope rep-
resents a good template for the design of a peptide antigen to
elicit neutralizing antibodies. In order to engineer a synthetic
immunogen capable of eliciting 4E10-like antibodies, a multistep
strategy is envisioned. The first step is to characterize the epitope
and determine its essential features. The core epitope has been
described as WF(D/N)IT (3), but the importance of the flanking
residues, especially at the C terminus, has been suggested from a
mutation study on the virus and from the recent crystal structure
of a peptide that included nine gp41 residues (residues 670 to
678) bound to the antibody (9, 40). Despite the wealth of mu-
tagenesis and structural data for 4E10, there have been no de-
tailed studies on synthetic peptides encompassing the 4E10
epitope. Therefore, the peptide length was first assessed to accu-
rately delimit the full extent of the epitope and an Ala scan was
performed on this expanded epitope to identify key amino acids
for binding to 4E10.
Synthetic peptides may elicit neutralizing antibodies only if
they bind in a conformation similar to that of the peptide
epitope of gp41 in the context of the virus. The crystallographic
structure suggests that the 4E10 epitope adopts a largely heli-
cal structure (9). Among the different techniques available to
increase the helicity of a peptide are the formation of con-
strained cyclic peptides and the substitution of the unnatural
* Corresponding author. Mailing address: Departments of Chemistry
and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines
Rd., CVN-6, La Jolla, CA 92037. Phone: (858) 784-7015. Fax: (858)
784-7319. E-mail: email@example.com.
† Supplemental material for this article may be found at http://jvi
amino acid 2-aminoisobutyric acid (Aib) (17, 23). Introduction
of a lactam bridge between a glutamic acid and a lysine at
positions i and i ? 4 (where i ? 4 represents the 4th amino acid
toward the C terminus compared to the amino acid in position
i), as well as i and i ? 3, is one of the best ways to constrain a
peptide and increase its helical content (35). We have recently
shown that thioether tethers are also useful for increasing the
helicity of a peptide (5). Peptides that encompassed the 4E10
epitope were designed and synthesized to incorporate helix-
promoting lactam bridges, thioether tethers, and Aib residues.
MATERIALS AND METHODS
Materials. Boc-amino acids, MBHA resin, and 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU) were obtained from
Peptides International (Louisville, KY). N,N-Diisopropylethylamine (DIEA),
fluoro tetramethylformamidinium hexafluorophosphate (TFFH), and anisole
were obtained from Sigma-Aldrich (St. Louis, MO). All solvents (high-perfor-
mance liquid chromatography [HPLC]-grade N,N-dimethylformamide [DMF],
dichloromethane, and acetonitrile) of high purity were purchased from Fisher.
Trifluoroacetic acid was obtained from Halocarbon Products (River Edge, NJ).
HF was purchased from Matheson Gas (Cucamonga, CA). The following re-
agents were obtained from the National Institutes of Health AIDS Research
and Reference Reagent Program: pNL4-3.Luc.R-E- (13) (contributed by N.
Landau), U87.CD4.CCR5 cells (4) (contributed by H. Deng and D. Littman),
JR-FL, TZM-bl cells (contributed by J. Kappes, X. Wu, and Tranzyme, Inc.)
(36), HIV-1SF162(contributed by J. Levy) (11), and HIV-1JR-CSF(contributed by
I. Chen) (8, 21). gp41 was purchased from Viral Therapeutics, Inc. (Ithaca,
N.Y.). HIV immunoglobulin (HIVIG) was provided by John Mascola (VRC,
Bethesda, Md.). HIV-1 neutralizing serum from patient FDA2 (31) was prepared
from blood drawn on 9 February 2005. 4E10 immunoglobulin G (IgG) was
generously provided by Hermann Katinger, Gabriela Stiegler, and Renate
CD. For the circular dichroism (CD) spectroscopy, an Aviv spectropolarimeter
model 203-02 was used, with cells of 0.1 cm in length, a wavelength step of 0.5
nm, and a bandwidth of 1.0 nm. One to three scans were reported. The exact
peptide concentrations were determined by UV measurements at 280 nm on a
Gison UV detector, model 116.
Peptide synthesis. The peptides were synthesized manually using solid-phase
peptide methodology on a C-terminal amide yielding MBHA resin, using in situ
neutralization cycles for Boc–solid-phase peptide synthesis (33). Aib was acti-
vated using 0.5 mmol Boc-Aib-OH, 0.5 mmol TFFH, and 0.7 ml DIEA in 1.5 ml
DMF for 15 min, 25°C. The activated amino acid was added to the deprotected
polypeptide resin without prior neutralization and coupled for 20 min. When
necessary, double couplings were performed. The N terminus of the peptides was
left unprotected. Solubilizing tails were introduced on the C-terminal end of the
peptide to allow easier synthesis of multiple compounds. Following chain assem-
bly, the peptides were cleaved from the resin with HF and 10% anisole for 1 h
at 0°C. The peptides were purified by HPLC. Analytical reversed-phase HPLC
was performed on a Rainin HPLC system equipped with a Vydac C18column (10
?m, 1.0 by 15 cm, flow rate of 1 ml/min). Preparative reversed-phase HPLC was
performed on Waters 4000 HPLC system using Vydac C18columns (10 ?m, 5.0
by 25 cm) and a Gilson UV detector. Linear gradients of acetonitrile in water–
0.1% trifluoroacetic acid were used to elute bound peptides. Peptides were
characterized by electrospray ionization mass spectrometry on an API-III triple
quadruple mass spectrometer (Sciex, Thornhill, Ontario, Canada). Peptide
masses were calculated from the experimental mass/charge (m/z) ratios from all
of the observed protonation states of a peptide by using MacSpec software
(Sciex). All observed peptide masses agreed with the calculated average masses
within 0.5 Da.
SPR. Surface plasmon resonance (SPR) experiments were performed using a
BIAcore 2000 instrument (Uppsala, Sweden).
Chip preparation. CM5 chips were coated with around 2,200 response units of
Fab 4E10. The carboxyl groups on the chip were activated with 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccin-
imide (NHS). Fifty micrograms of Fab (prepared as described in reference 9) was
diluted in 10 mM sodium acetate, pH 4.5; a flow rate of 5 ?l/min was used.
Unreacted carboxyl groups were blocked with 1 M ethanolamine at pH 8.5. The
control was treated in the same fashion without any antibody present.
SPR measurements. Different amounts of free peptides were then passed over
the surfaces at 30 or 50 ?l/min for 2 min. Regeneration was done in HPS-EP
buffer with 0.25 NaCl (BIAcore) in 10 min. The amount of salt was increased
compared to that in the commercial buffer to reduce the nonspecific binding.
Data evaluation. For data evaluation, the BIAevaluation software was used.
RI and Rmaxwere controlled, and double referencing was done (0 concentration
and start point). Analyses were performed to achieve the best curve fitting and
ELISAs. Fifty percent inhibitory concentrations (IC50s) were determined by
competitive enzyme-linked immunosorbent assay (ELISA) using a constant con-
centration of biotinylated peptide and IgG with a variable concentration of gp41
peptides. Microwells were coated overnight at 4°C with 50 ?l phosphate-buffered
saline (PBS) containing neutravidin (Pierce; 4 ?g/ml). Wells were washed twice
with PBS containing 0.05% Tween 20 and blocked with 4% nonfat dry milk in
PBS for 45 min at 37°C. Meanwhile, a mixture of a biotinylated 4E10-epitope
peptide, SLWNWFDITNWLWRRK(biotin)-NH2, (20 nM), IgG 4E10 (0.2 nM),
and the competing peptide analogue (threefold dilution series starting at 10 ?M)
in 0.4% nonfat dry milk, 0.02% Tween, and PBS was incubated in a separate
96-well plate at 37°C for 2 h. After washing the blocked plate, the mixture of
4E10, biotinylated peptide, and competing peptide was added to the wells. After
20 min at room temperature, the wells were washed five times, and a 1:500
dilution of goat anti-human IgG F(ab?)2–horseradish peroxidase conjugate
(Pierce) was added. Following incubation at room temperature for 40 min, the
wells were washed five times, and developed by adding 50 ?l of tetramethylben-
zidine (TMB) solution (Pierce) according to the manufacturer’s instructions.
After ?20 min, wells containing TMB solution were stopped by adding 50 ?l of
H2SO4(2 M), and the optical density at 450 nm was read on a microplate reader
(Molecular Devices). The concentration of competitor peptide corresponding to
a half-maximal signal (IC50) was determined by interpolation of the resulting
binding curve. Each peptide competitor was tested in duplicate in at least two
HIV-1 neutralization assays. Neutralization assays were performed in two
different formats. In the first, replication-competent HIV-1SF162was assayed for
neutralization using TZM-bl cells as indicator cells (36). Alternatively, a
pseudotype assay was used in which recombinant HIV-1JR-CSFvirions, compe-
tent for a single round of infection, were generated using the luciferase reporter
plasmid pNL4-3.Luc.R-E-, as described previously (13, 41), and the pseudovirus
was assayed for neutralization using U87.CD4.CCR5 cells as target cells (4). In
all cases, the competitor peptide NWFDITNWLWYIKKKK-NH2(60 ?g/ml)
and different concentrations of IgG 4E10 were preincubated for 30 min at 37°C,
and then this mixture was added (1:1 by volume) to HIV-1, and the resulting
mixture was incubated for a further 1 h at 37°C. The mixture of peptide, 4E10,
and HIV-1 was then added (1:1 by volume) to the target cells, and the assay was
developed using luciferase reagent (Promega) following a 48- to 72-h incubation
at 37°C. The degree of virus neutralization was determined as a percentage
reduction of viral infectivity against an antibody-free control. All experiments
were performed in triplicate and repeated at least twice with similar results.
Characterization of the full 4E10 peptide epitope. In order
to identify the minimal gp41 peptide sequence that binds
tightly to 4E10, a series of peptides were synthesized and 4E10
binding was measured by ELISA. Previous studies had identi-
fied residues NWFDIT (gp41 residues 671 to 676) to be an
important part of the core 4E10 epitope (34, 42). The impor-
tance of W680 had also been shown from an alanine scan of
the gp41 membrane-proximal external region (MPER) on the
virus, using 4E10 neutralization as a readout, and was also
suggested from analysis of the crystal structure of an 13-amino-
acid peptide (named “KGND”, including gp41 residues 670 to
678 bound to 4E10) (9, 40). Therefore, an extended sequence,
NWFDITNWLW, corresponding to gp41 residues 671 to 680
was selected as a starting point to identify the full linear
epitope. The resulting peptide, NWFDITNWLWKKKK-NH2,
had an IC50of 40 nM (Table 1, 84-1). A C-terminal polylysine
tail was introduced to improve peptide solubility (?2 mg/ml in
PBS was attained for most analogs used in these studies). The
polylysine tail is not expected to make direct interactions with
VOL. 80, 2006 CONSTRAINED PEPTIDES AS PROBES OF HIV-1 ANTIBODY 4E10 1681
the 4E10 antibody, consistent with the poor binding of WNW
FDITNKKKK-NH2(178-1, Table 1).
The extent of the 4E10 peptide epitope was further charac-
terized by extending this sequence toward the N and C termini.
N-terminal extensions of the epitope did not improve 4E10
binding (Table 1, 84-1 compared to 84-2 and 84-4). C-terminal
extension of the sequence up to the transmembrane domain
(residue 683) increased 4E10 binding by fourfold with respect
to our starting point (Table 1, 84-1 compared to 94-1). These
results suggest that residues 671 to 683 of gp41 (NWFDIT
NWLWYIK) represent the shortest linear epitope with opti-
mal affinity for 4E10. A peptide encompassing this sequence
with a solubilizing lysine tail, NWFDITNWLWYIKKKK-NH2,
had an IC50of 10 nM, an improvement of 4-fold over our starting
peptide and an improvement over 1,000-fold compared to
KGND, the 13-mer used in the crystal analysis (Table 1).
Alanine scan. The importance of individual amino acid side
chains can be assessed by performing an alanine scan. Alanine
was individually substituted for each amino acid in the opti-
mized epitope (residues 671 to 683). The effects of these mu-
tations on the IC50are shown in Fig. 1A. Mutations at W672,
F673, and T676 resulted in a major decrease in binding to the
4E10 antibody (over 1,000-fold) and confirm that these three
residues are crucial for peptide recognition by 4E10. The next
major increase in IC50was observed when L679 was mutated to
alanine. The importance of this residue had not been predicted
in prior reports. Four other residues (N671, D674, I675, and
W680) also showed a decrease in binding of 20- to 30-fold
when alanine substitutions were performed. The other residues
in the sequence could be replaced with alanine without any
major decrease in 4E10 binding (fivefold or less).
Introduction of constraints. A recent structural study of the
4E10-peptide complex showed that the bound conformation of
the peptide is helical (9). Therefore, helix-inducing constraints
were introduced, including Aib residues and side-chain tethers
(Table 2). Peptides in which “WF” was not included in the
cyclic tether showed substantially increased binding to 4E10,
indicating that these particular constraints on “WF” interfere
with binding (Table 2, 74-2). Constraints in the center and C
terminus resulted in peptides with a tighter binding to 4E10
compared to peptides with a constraint found in the N termi-
nus, suggesting that increasing the helical character in the
central region is favorable for 4E10 binding (Table 2, 104-2).
These results are consistent with the crystal structure of
“KGND” bound to the antibody in which the helix begins to
“unwind” at residue W672, where the N terminus of the ?-helix
abuts the antibody combining site (9). Tightly binding peptides
(IC50of 10 nM) were obtained that incorporated either Aib
residues or thioether tethers.
Circular dichroism spectroscopy. To determine the relative
helicity of the gp41 peptide analogs, each one was analyzed in
solution by CD spectroscopy. The tightest-binding peptides
were all helical, with minima close to 207 and 222 nm. How-
ever, a further increase in helicity did not always result in an
increase in binding: 94-1 is more helical than 84-1 and has a
smaller IC50(10 nM versus 40 nM, Tables 1 and 2); however,
119, which is more helical than 94-1, had the same IC50(10
nM, Table 2; Fig. 2, right panel). Nevertheless, the imposed
constraints increased helicity in solution without diminishing
FIG. 1. (A) Effects of Ala substitutions (along the epitope) on
4E10 binding to synthetic peptides. The bars represent the ratio log
(IC50_peptide reference/IC50_mutant). (B) Helical wheel representa-
tion of gp41 (671 to 683). The key binding residues for 4E10 are
underlined and are all found on the same side of the helix. The values
for the log (IC50_mutant/IC50_peptide reference) of W672, F673, and
T675 represent a minimum, since the IC50increased by a factor of
greater than 1,000 when Ala was substituted for those amino acids.
IC50s were measured in two sets of experiments. For W672, F673, I675,
T676, and W680, Ala substitutions were performed on the 14-mer
NWFDITNWLWKKKK-NH2(IC50? 40 nM). For the rest, the sub-
stitutions were performed on the 17-mer SLWNWFDITNWLWYI
KKKK-NH2(IC50? 10 nM).
TABLE 1. Amino acid sequences and 4E10 binding data (IC50and Kd)
of some of the unconstrained peptide analogs
aThe amino acids shown in bold belong to the native sequence. O represents
bND, not determined.
1682BRUNEL ET AL.J. VIROL.
4E10 binding. Slightly shorter, structurally constrained pep-
tides with tight binding to 4E10 (IC50? 10 nM) were also
identified (Table 2, compounds 102-1 and 104-2).
In general, the constrained peptides that adopt a helical
conformation in solution bind with greater affinity to 4E10
than similar peptides that are poorly structured in solution.
In one example, a side-chain-tethered peptide, 104-2 [NWFc
(CITO)WLWKKKK-NH2], was found to have an increased
helical content relative to its unconstrained counterpart, as
determined by the appearance of two minima in the CD spec-
trum (gray triangles, Fig. 2, left panel). [c(CITO) indicates the
presence of a bridge linking the side chains of cysteine and
ornithine.] The binding affinity of the cyclic peptide to 4E10
was improved by fourfold (104-1 in Table 1 compared to 104-2
in Table 2). Note that the extended native sequence is also
quite helical (squares, Fig. 2, right panel). The CD spectra of
all the other peptides mentioned in this article can be found in
the supplemental material.
SPR. The affinities of several peptide analogues were mea-
sured using SPR to validate the ELISA. Three peptides were
picked to represent a range of affinities on related peptides.
For each peptide, Kdvalues and on (kon) and off (koff) rates
were determined: 84-1 (IC50? 40 nM), Kd? 100 nM, kon?
1.49 ? 105M?1s?1, and koff? 0.0149 s?1; 74-2 (IC50? 230
nM), Kd? 277 nM, kon? 1.75 ? 105M?1s?1, and koff?
0.0485 s?1; and 104-2 (IC50? 10 nM), Kd? 17 nM, kon? 2.02 ?
105M?1s?1, and koff? 0.00336 s?1. The Kdvalues obtained
from the Biacore analysis were in good agreement with the
ELISA results and were all within a factor of 1.2 to 2.5 higher
than the corresponding IC50values, as determined by ELISA
(Tables 1 and 2). The on rates of the three peptides are very
similar, as is typically observed for the structurally similar pep-
tide analogs (1). In contrast, the off rates vary by an order of
magnitude, consistent with the various stabilities of the bound
peptides as reflected by a well-positioned thioether tether
(104-2) versus a poorly positioned thioether tether (74-2) com-
pared to the linear peptide 84-1. The Kdof the tightest-binding
linear peptide (94-1; IC50, 10 nM) was also analyzed by SPR
and was found to be 20 nM.
The affinity-optimized native sequence, as well as the se-
quences of several of the constrained peptides, all bind the
4E10 neutralizing antibody with affinities in the nanomolar
range (Kd, ?20 nM). Their IC50s were determined by ELISA
to be around 10 nM. Note that an IC50of 0.25 ?g/ml was
determined for recombinant gp41 (residues 541 to 682 accord-
ing to HxB2) (Viral Therapeutics, Inc., Ithaca, NY), which, if
we assume gp41 has an average molecular mass of 25 kDa and
is largely monomeric in solution, is equal to an IC50of around
10 nM (data not shown). However, this value can only be
considered a rough approximation and may differ substantially
if gp41 is not monomeric in solution.
Effect of the peptide on HIV-1 neutralization by 4E10. To
further investigate the interaction of peptide analogs and 4E10,
the inhibitory effect of the best analogs on neutralization by
4E10 was assessed. Peptide 94-1 [NWFDITNWLWYIKKKK-
NH2] produced the most favorable and reproducible inhibition
of 4E10 neutralization in initial experiments. This peptide
could block the neutralization by 4E10 of replication-compe-
tent primary isolates, SF162 and JRCSF, at 30 ?g/ml (Fig. 3A).
The peptide also blocks neutralization under conditions in
which normal serum was spiked with 4E10 (Fig. 3B). Under
similar conditions, this peptide does not block neutralization
by polyclonal IgG from HIV-1-infected donors (HIVIG) or by
FIG. 2. CD spectra of free 4E10-epitope peptides with or without helix-promoting constraints. The presence of two minima is consistent with
a helical conformation. An acyclic compound (black circles) is compared to its cyclic analog (gray triangles) (left panel). Two native linear
sequences of the 4E10 epitope are compared to an Aib-containing analog (black triangles) (right panel). deg., degrees.
TABLE 2. Amino acid sequences and 4E10 binding data
(IC50and Kd) of some of the constrained analogs
aThe amino acids shown in bold belong to the native sequence. B indicates
Aib. The amino acids underlined are in a cyclic conformation.
bND, not determined.
VOL. 80, 2006 CONSTRAINED PEPTIDES AS PROBES OF HIV-1 ANTIBODY 4E101683
the reference serum, FDA2 (Fig. 3B). These results show that
the peptide interacts with the 4E10 antibody, preventing it
from interacting with (i.e., neutralizing) the virus, but the
4E10-like antibodies are not present in appreciable titers in the
polyclonal and serum samples tested. We noticed in our initial
experiments that some peptides enhanced infectivity of the
virus, whereas others inhibited it, but such effects were typically
nonspecific, as a vesicular stomatitis virus G-pseudotyped virus
was similarly affected (data not shown). The reasons for these
observations are unknown but may be due to differences in cell
viability, membrane perturbation, or other properties of the
While most of the surface of gp41 is thought to be hidden
within the native trimer prior to fusion, some epitopes of gp41
appear to be somewhat accessible during, and more so follow-
ing, receptor activation, when gp41 switches from the native
configuration through a pre-hairpin intermediate to the post-
fusion structure (2, 14, 16, 32). Specifically, the MPER of gp41
encompasses the epitopes for three neutralizing antibodies
(4E10, 2F5, and Z13). However, immunogens incorporating
MPER sequences have failed to elicit antibodies with the
breadth and potency associated with these existing neutralizing
antibodies (2, 12, 15, 19, 22, 24, 27). One explanation for the
failure of at least some of these immunogens is that the peptide
epitopes have been minimized to the extent that they adopt a
largely unstructured conformation in solution and that immu-
nogens based on extended MPER sequences or those with
greater constraints should be better candidates (2). Alterna-
tively, it has been proposed that to be effective immunogens,
MPER sequences may require a membrane context, since the
binding affinity of 4E10 and 2F5 to gp41 peptides increases in
the presence of a membrane (28). A final concern has been
raised by Haynes et al., who suggest that 2F5 and 4E10 cross-
react with autoantigens, such as cardiolipin, and that MPER
epitopes could mimic autoantibody epitopes (18). In this case,
the B cells making antibodies to the MPER would be clonally
deleted or suppressed, and this would explain the failure of
MPER immunogens. An alternative explanation of any cross-
reactivity observed, at least for 4E10, is that it arises from the
highly hydrophobic nature of the binding site of this antibody
(7). In any case, it is clear, however, that a detailed character-
ization of the 4E10 peptide epitope and the synthesis of pep-
tide antigens that mimic the structure of this epitope are valu-
able steps toward the use of the design of immunogens eliciting
antibodies to the MPER.
We decided to focus on the human monoclonal antibody
4E10 as it is the most broadly neutralizing antibody described
to date. A recent crystallographic study shows that the peptide
KGWNWFDITNWGK (called “KGND”) adopts a largely he-
FIG. 3. Neutralization experiments with the peptide representing
the newly optimized epitope and its solubilizing tail, NWFDITNWL
WYIKKKK-NH2. (A) Ability of peptide NWFDITNWLWYIKKKK-
NH2to block neutralization of HIV-1 by 4E10. Replication-competent
HIV-1 strains (SF162 and JR-CSF) produced in human peripheral
blood mononuclear cells were assayed for neutralization by 4E10 (100
?g/ml) in TZM-bl cells, in the presence (white bars) or absence (black
bars) of an excess of peptide. (B) Effect of peptide NWFDITNWL
WYIKKKK-NH2on neutralization of HIV-1 by polyclonal antibodies
and sera. HIV-1JR-FL, pseudotyped using the pNL4-3.Luc reporter
plasmid, was assayed for neutralization using pooled polyclonal IgG
from HIV-1-seropositive individuals (HIVIG), broadly neutralizing
serum from the FDA2 individual, and normal human serum spiked
with 4E10 at 200 ?g/ml in the undiluted serum. Neutralization assays
were performed using U87.CD4.CCR5 cells as target cells, in the pres-
ence (open symbols) or absence (closed symbols) of peptide NWFDIT
NWLWYIKKKK-NH2(Note that the zero point in serum dilution cor-
responds to 30 ?g/ml of 94-1 being present.)
1684BRUNEL ET AL. J. VIROL.
lical structure with all of the crucial amino acids for binding
being presented on the same side of the helix (9) We refer to
the residues that are not involved in binding to the antibody as
the “nonneutralizing face,” in keeping with the same terminol-
ogy used for the trimeric envelope spike (38) (Fig. 1B). The
4E10 epitope is not trivial to mimic because it is not a perfect
?-helix throughout its entire length and the crucial residues
“WF” are in a 310-helical structure, which is frequently ob-
served to terminate ?-helical structures. Thus, designing a per-
fect ?-helix might not generate the optimal candidate for im-
The ability of a peptide to elicit a strong immune response is
not predictive of its ability to elicit neutralizing antibodies. This
problem has been encountered for 2F5 (12, 15, 19, 22, 24, 27).
Furthermore, the affinity of an antibody for a particular anti-
gen (antigenicity) is not necessarily predictive of the ability of
the same antigen to elicit that antibody (immunogenicity). To
develop an effective antigen, we envision a multistep strategy.
Initially, the particular epitope is characterized: in this case, by
first identifying the length of the peptide that gives the tightest
binding to the antibody and then performing alanine mutations
to find the key amino acids. The next stage consists of restrict-
ing the peptide conformation to the one adopted when bound
to the neutralizing antibody (in this case, a helical conforma-
tion). This step has been satisfactorily achieved in the present
study. The last stage will consist of the replacement of unnec-
essary parts with less immunogenic substituents to mask the
“nonneutralizing face” without perturbing the constrained
conformation. This final step will ensure that only the side of
the helix which is involved in the binding to the antibody will be
available to the immune system (Fig. 4). Masking the “non-
neutralizing” face is a principle that has been suggested to
focus the immune response (28, 30). Finer modifications will
be evaluated iteratively and empirically in subsequent stages.
In this study, we have identified the optimal length of the
peptide epitope as NWFDITNWLWYIK (residues 671 to
683). A peptide containing this epitope and a solubilizing tail
has an IC50of 10 nM in peptide competition experiments and
a Kdof 20 nM (as measured via BIAcore) (Table 1). This
peptide also blocked neutralization of different HIV-1 strains
In order to identify permissive sites for further modification
to the 4E10 epitope, an Ala scan was performed. Alanine
substitution at residues W672, F673, and T676 resulted in a
major loss of binding to 4E10 (over 1,000-fold decrease) (Fig.
1). Because substitution in these positions also slightly in-
creased the helicity (CD; see the supplemental material), the
loss of binding does not then appear to result from a loss of
helical structure. These binding results are in agreement with
the 4E10 crystal structure in complex with the “KGND” pep-
tide (9), where W672, F673, and T676 make intimate contacts
with the antibody. Mutation of these amino acids on the virus
also decreased neutralization of the mutant virus by 4E10 (40).
Taken as a whole, these studies confirm that W672, F673, and
T676 are important components of the 4E10 epitope.
Surprisingly, mutation of L679 to alanine resulted in a major
decrease in 4E10 binding (70-fold). The importance of this
residue could not have been foreseen from the crystal structure
since L679 was replaced with a glycine spacer in the 13-mer
used in the crystal data (9). The mutation L679A resulted in a
small decrease in the helical character of the peptide, but not
enough to account for the observed 70-fold loss in binding.
Therefore, we believe that L679 makes direct contact with the
antibody. During the Ala scan on the virus, L679 was not found
FIG. 4. Schematic of the vaccine design process where constraints are introduced. (a) The peptides are constrained to a helix conformation via
the introduction of an Aib or tether constraint. (b) The “nonneutralizing face” is blocked with the introduction of nonimmunogenic bulk so
antibody is preferentially elicited against the neutralizing face.
VOL. 80, 2006 CONSTRAINED PEPTIDES AS PROBES OF HIV-1 ANTIBODY 4E101685
to be critical, as the L679A virus could still be neutralized. The
differences in the effect of Ala substitutions on peptide/affinity
versus HIV-1 neutralization by 4E10 are discussed below (40).
The substitutions I675A and W680A also resulted in major
increases in IC50(20- to 30-fold). The importance of I675 is
predicted from the crystal structure, where it was found that
I675 makes contact with the antibody, but less than W672,
F673, and T676 (9). The I675 substitution also resulted in a
slight decrease in helical character, which could have affected
the 4E10 binding. W680A resulted in a pronounced loss of
binding of the peptide to 4E10, while the helical character was
improved. The importance of W680 had been seen in the
mutation study performed on the virus, as the W680A muta-
tion decreased neutralization of the mutant virus by 4E10 (40)
and was also suggested from the crystallographic analysis, even
though the tryptophan had been replaced by a lysine to obtain
a soluble peptide for crystallization (9). Alanine substitutions
on the “nonneutralizing face” usually did not result in major
increases in IC50, with the exception of D674A. The alanine
substitutions N671A and D674A resulted in a disruption of the
peptide conformation (CD; see the supplemental material).
These two residues do not make contact with 4E10 in the
crystal structure (9); therefore, they apparently play an impor-
tant role in stabilizing the structure of the peptide in a helical
conformation. Also, the Ala scan on the virus shows that N671
and D674 are not critical for neutralization (40). Similarly,
mutations of I682 and K683 strongly decrease the helical con-
tent of the peptide (CD; see the supplemental material), which
may explain the lower affinity of the respective mutants. These
residues also play an important role in stabilizing the peptide
structure, but probably do not make contact with the antibody.
Finally, N677, W678, and Y681 could be mutated to alanine
with no major effect on the binding affinity to 4E10 (increase of
less than twofold) or on the peptide structure.
The Ala scan allowed us to refine the synthetic peptide epitope
of 4E10 as NWFDITnwLWyIK, with the uppercase letters as
important residues (among them W, F, T, and L are the major
residues) and the lowercase letters as replaceable ones. We
believe that appropriate modifications of residues as N677 and
W678 (found on the nonneutralizing face of the helix) should
not affect binding to 4E10 but could result in a reduction of the
immunogenicity of this side of the helix. The importance of
some of the residues concurs with mutagenesis experiments
performed on the virus, in which neutralization resistance oc-
curred with the substitutions W672A, F673A, and W680A (40).
However, in general, these results show how peptides in solu-
tion may behave quite differently from the corresponding re-
gion on a folded protein that is anchored to a membrane. In
our study, both faces of the helix are exposed to water, whereas
the “nonneutralizing” face on the virus may be interacting with
neighboring protomers of gp41 or gp120 within the trimer or
with the membrane. This difference in the surrounding envi-
ronment could explain the differences between the Ala scans
on the peptide and those on the virus. Moreover, the Ala
substitutions in the viral protein may affect the entry kinetics of
the virus, causing enhanced susceptibility of the virus to 4E10
without affecting the intrinsic affinity to the membrane-proxi-
mal external region epitope.
The next step of our strategy focused on limiting the con-
formational diversity of the peptides by designing analogs that
are constrained to adopt a conformation in solution similar to
that of the peptide bound to 4E10. In the crystal structure, the
4E10 epitope peptide is in a largely helical conformation. Pep-
tides derived from the native gp41 sequence are generally
helical in PBS buffer (Fig. 2, squares, right panel). In order to
reduce alternative peptide conformations, constraints were in-
troduced to further enhance this helical propensity through the
use of cyclothioethers, lactams, and reversed lactam bridges, as
well as Aib-containing analogs. The presence of a helical con-
formation is generally associated with strong 4E10 binding.
We introduced constraints closer to the N terminus of the
sequence, initially forming thioether tethers (residues 670 to
674 or 671 to 674). These peptides did not show significant
binding to the antibody. When we moved the position of cy-
clization toward the center or the C terminus to constrain
residues 674 to 677 or 674 to 678, we saw an increase in
binding: the cyclic ether formed between residues 674 and 677
is among our best derivatives (Table 2). This result is in agree-
ment with the crystal structure, as the peptide is more ?-helical
toward the center and the C terminus. The incompatibility of
N-terminal tethers may be due to a steric clash with the 4E10
binding pocket or the transition of the ?-helix to a 310helix at
the N terminus (9).
In summary, cyclic and acyclic analogs (native or Aib con-
taining) were identified in which the tight binding to 4E10 (10
nM) was maintained (Table 2) and yet the possible backbone
conformations adopted by the different analogs were re-
stricted. Although, in some cases, further enhancement of he-
licity or structure did not increase 4E10 binding, we anticipate
that the more rigid peptides will be more specific immunogens.
Compatibility of an Aib substitution with tight 4E10 binding is
very promising for the use of such peptides in the design of a
vaccine. Not only does the presence of an Aib residue increase
the helicity, it also destabilizes alternative conformations. Such
stability may be particularly useful in the presence of denatur-
ing adjuvants. In addition, Aib introduces a minimal structural
modification, reducing the chances of directing an immune
response to the constraint. Therefore, the sequences described
here would appear to be useful candidates for immunization
studies. The best analogs from each series (an Aib-containing
peptide, a lactam, and a thioether) are now being assessed in
We thank Peter Wright and Linda Tennant, TSRI, for assistance
with CD and Laure Jason-Moller, BIAcore, for advice on BIAcore. We
are thankful to Hermann Katinger, Gabriela Stiegler, and Renate
Kunert, Vienna, for providing us with 4E10 IgG.
We acknowledge support from the American Foundation for AIDS
Research (to F.M.B. and R.M.F.C.), the Elizabeth Glaser Pediatrics
AIDS Foundation (to M.B.Z.), the NIH (AI 058725 to M.B.Z, AI
33292 to D.R.B, GM46192 to I.A.W., and MH062261 to P.E.D.), the
Neutralizing Antibody Consortium of the International AIDS Vaccine
Initiative, the Pendleton Trust, and the Skaggs Institute for Chemical
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