Remodeling of dynamic structures of HIV-1 envelope proteins leads to synthetic antigen molecules inducing neutralizing antibodies

Article abstract of DOI:10.1021/bc900502z

A synthetic antigen targeting membrane-fusion mechanism of HIV-1 has a newly designed template with C3-symmetric linkers mimicking N36 trimeric form. The antiserum produced by immunization of the N36 trimeric form antigen showed structural preference in b

Full text of DOI:10.1021/bc900502z

Bioconjugate Chem. 2010, 21, 709–714
709
Remodeling of Dynamic Structures of HIV-1 Envelope Proteins Leads to
Synthetic Antigen Molecules Inducing Neutralizing Antibodies
†
,†
‡
†
†
†
Toru Nakahara, Wataru Nomura,* Kenji Ohba, Aki Ohya, Tomohiro Tanaka, Chie Hashimoto,
†
‡
‡
,†
Tetsuo Narumi, Tsutomu Murakami, Naoki Yamamoto, and Hirokazu Tamamura*
Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan, and AIDS Research Center, National Institute of Infectious Diseases,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Received November 16, 2009; Revised Manuscript Received February 28, 2010
A synthetic antigen targeting membrane-fusion mechanism of HIV-1 has a newly designed template with C3-
symmetric linkers mimicking N36 trimeric form. The antiserum produced by immunization of the N36 trimeric
form antigen showed structural preference in binding to N36 trimer and stronger inhibitory activity against HIV-1
infection than the N36 monomer. Our results suggest an effective strategy of HIV vaccine design based on a
relationship to the native structure of proteins involved in HIV fusion mechanisms.
INTRODUCTION
C-terminal peptides, although its biological advantages have not
been determined (15). The mimicry can be estimated using the
broadly neutralizing mAbs; suitable mimetics will bind neutral-
izing mAbs efficiently, but they will bind non-neutralizing mAbs
poorly. In the present study, we designed and synthesized a
novel three-helical bundle mimetic, which corresponds to the
trimeric form of N36. We investigated whether mice immunized
with the equivalent trimeric form of N36 mimetic can produce
antibodies with stronger binding affinity for N36 trimer than
for N36 monomer. This approach demonstrates the possibility
of producing structure-specific antibodies by immunization of
synthetic antigens corresponding to the natural form of viral
proteins.
Antibody-based therapy is one of the promising treatments
for AIDS. In recent years, AIDS antibodies have been produced
by immunization (1) and by de novo engineering of monoclonal
antibodies (mAb) with molecular evolution tactics such as phage
display (2). Despite enormous efforts, however, only a limited
number of highly and broadly HIV-neutralizing human mAbs
have been isolated and characterized. These antibodies include
gp41 Abs, 2F5 (3-6) and 4E10 (5-7), and gp120 Abs, 2G12
(8) and b12 (9). gp41 is a transmembrane envelope glycoprotein,
which is divided into an endodomain and an ectodomain by
the transmembrane region; the latter contains a hydrophobic
amino-terminal fusion peptide, followed by amino-terminal and
carboxy-terminal leucine/isoleucine heptad repeat domains with
helical structures (HR1 and HR2, respectively). In the membrane
fusion process of HIV-1, these subunits form a “pre-bundle”
complex. The HR1 and HR2 regions are termed the N-terminal
helix (N36) and C-terminal helix (C34), respectively. These
helices form a six-helical bundle consisting of a central parallel
trimeric coiled-coil of N36 surrounded by C34 in an antiparallel
hairpin fashion. In design of immunogens that elicit broadly
neutralizing antibodies, a useful strategy is to produce molecules
that mimic the natural trimer on the virion surface. Previous
studies show that these molecules could be proteins expressed
as a recombinant form or on the surface of particles such as
pseudovirions or proteoliposomes (10-12). The X-ray crystal-
lographic study of gp41 shows that the distances between any
two residues at the N-terminus of N-region are almost equal at
approximately 10 Å (Figure 1A). A chemically synthetic
template could be useful in connection with the design of a
peptidomimetic corresponding to the native structure of gp41.
To date, several gp41 mimetics have been synthesized as
inhibitors or antigens and subjected to inhibition or neutralization
assays (13-16). However, the templates for assembly of these
helical peptides contain branched peptide linkers, which are not
exactly equivalent in length (14). The N-terminal peptides
constrained by another threefold linker showed high affinity for
EXPERIMENTAL PROCEDURES
Conjugation of N36REGC and the Template to Pro-
duce triN36e. Compound 6 (100 µg, 0.174 µmol) and N36REGC
(3.4 mg, 0.574 µmol) were dissolved in a mixture of 300 µL of
200 mM acetate buffer (pH 5.2) and 300 µL of TFE under a
nitrogen atmosphere, then TCEP ·HCl was added. The reaction
was stirred for 72 h at room temperature and monitored by
HPLC. The ligation product (triN36e) was separated as an HPLC
peak and was characterized by ESI-TOF-MS, m/z calcd for
C690H1160N226O201S3 15933.1, found 15933.8. The purification
was performed by reverse phase HPLC (YMC-Pack ODS-A
column, 10 × 250 mm). Elution was carried out with a 40-50%
linear gradient of acetonitrile (0.1% TFA) over 50 min. Purified
triN36e, obtained in 16% yield, was identified by ESI-TOF-
MS. The detailed synthesis of peptides is described in the
Supporting Information (SI).
CD Spectra. CD measurements were performed with a J-720
circular dichroism spectrolarimeter equipped with a thermoregu-
lator (JASCO). The wavelength dependence of molar ellipticity
[θ] was monitored at 25 °C from 190 to 250 nm. Peptides were
dissolved in 20 mM acetate buffer (pH 4.0) containing 40%
MeOH (23, 24). The experimental helicity was calculated as
reported previously (17-19).
Immunization and Sample Collection. Six-week-old male
BALB/c mice were purchased from Sankyo Laboratory Service
Corp. (Tokyo, Japan) and maintained under specific pathogen-
free conditions in an animal facility. The experimental protocol
was approved by the ethical review committee of Tokyo Medical
and Dental University. Freund incomplete adjuvant and PBS
*
To whom correspondence should be addressed. E-mail:
nomura.mr@tmd.ac.jp; tamamura.mr@tmd.ac.jp. phone: +81-3-5280-
8
036, fax: +81-3-5280-8039.
†
Tokyo Medical and Dental University.
National Institute of Infectious Diseases.
‡
1
0.1021/bc900502z  2010 American Chemical Society
Published on Web 04/01/2010

710 Bioconjugate Chem., Vol. 21, No. 4, 2010
Nakahara et al.
Figure 1. (A) Distances between hydrogen atoms for hydroxyl groups in N-terminal serine residues of N36 helices in trimeric form. The distances
were evaluated by PyMOL (21). (B) Cartoon presentation of each N36 derived peptide, N36REGC. (C) Design of a C3-symmetric template. The
amino acid residues are described in single letters. (D) Conjugated structure of trimeric N36 after thiazolidine ligation.
were purchased from Wako Pure Chemical Industries (Osaka,
Japan). DMSO (endotoxin free) was purchased from Sigma-
Aldrich (St. Louis, MO).
Carlsbad, CA) followed by changing medium at 12 h after
transfection. At 48 h after changing medium, the supernatant
was collected, passed through a 0.45 µm filter, and stored at
-80 °C as HIV-1NL4-3 strain before use. For titration, MT-4
cells were infected with serially 3-fold diluted virus from 1/10
to 1/196830, and cultured for 7 days. HIV-1 p24 levels in
supernatants were measured, and then the titer of virus solution
was calculated.
All mice were bled one week before immunization. One
hundred micrograms of antigen was dissolved in 1 µL of DMSO.
The solution was mixed with 50 µL of PBS and 50 µL of Freund
incomplete adjuvant. The mixture was injected subcutaneously
under anesthesia on days 0, 14, 28, 42, and 58. Mice were bled
on days 21, 35, 49, and 65. Serum was separated by centrifuga-
tion (15 000 rpm) at 4 °C for 15 min and inactivated at 56 °C
for 30 min. Sera were stored at -80 °C before use.
Serum Titer ELISA. Tween-20 (polyoxyethylene (20)
sorbitan monolaurate) and hydrogen peroxide (30%) were
purchased from Wako. ABTS (2,2-azino-bis(3-ethylbenzothia-
zoline-6-sulfonic acid) diammonium salt) was purchased from
Sigma-Aldrich. Antimouse IgG (H+L)(goat)-HRP was pur-
chased from EMD Chemicals (San Diego, CA). Ninty-six-well
microplates were coated with 25 µL of a synthetic peptide at
Anti-HIV Assay. Virus was prepared as described above
except that the transfection of pNL4-3 was performed by the
calcium phosphate method. Anti-HIV-1 activity was determined
on the basis of protection against HIV-1-induced cytopathoge-
nicity in MT-4 cells. Various concentrations of AZT, N36RE,
and triN36e (The starting concentrations are 100, 10, and 1 µM,
respectively) were added to HIV-1-infected MT-4 cells (MOI
) 0.01) by 2-fold serial dilution and placed in wells of a flat-
4
bottomed microtiter plate (2.0 × 10 cells/well). After 5 days’
2
incubation at 37 °C in a CO incubator, the number of viable
1
0 µg/mL in PBS at 4 °C for overnight. The coated plates were
cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method (EC50). Cytotox-
icity of compounds was determined on the basis of viability of
mock-infected cells using the MTT method (CC50). Each
experiment was performed three times independently.
washed 10 times with deionized water and blocked with 150
µL of blocking buffer (0.02% PBST, PBS with 0.02% Tween
2
0, containing 5% skim milk) at 37 °C for 1 h. The plates were
washed with deionized water 10 times. Mice sera were diluted
in 0.02% PBST with 1% skim milk, and 50 µL of 2-fold serial
dilutions of sera from 1/200 to 1/102400 were added to the wells
and allowed to incubate at 37 °C for 2 h. The plates were washed
5
Neutralizing Assay. MT4-cells (1 × 10 cells/100 µL) were
incubated in 100 µL medium containing 10 µL sera from
immunized or preimmunized mice for 1 h at 37 °C, then
pretreated MT-4 cells were infected with HIV-1NL4-3 (MOI )
0.05). At 3 days after infection, cells were collected by
centrifuge at 4000 rpm for 10 min at 4 °C. After discarding
supernatant, pellets were lysed with 30 µL of lysis buffer (50
mM Tris ·HCl (pH 7.5), 150 mM NaCl, 1% NP-40), then 30
µL of 2 × SDS buffer (125 mM Tris ·HCl (pH 6.8), 4% SDS,
20% glycerol, 10% 2-ME, 0.004% BPB) were added and boiled
for 10 min. The samples (5 µL) were subjected to SDS-page to
perform Western blotting. The HIV-1 gag p24 was detected by
using Western lightning ECL kit (PerkinElmer, MA) according
to manufacturer’s instruction after treatment of HIV-1 p24
10 times with deionized water. Twenty-five microliters of HRP-
conjugated antimouse IgG, diluted 1:2000 in 0.02% PBST, was
added to each well. After 45 min incubation, the plates were
washed 10 times and 25 µL of HRP substrate, prepared by
dissolving 10 mg ABTS to 200 µLof HRP staining buffersa
mixture of 0.5 M citrate buffer (pH 4.0, 1 mL), H
2 2
O (3 µL),
and H O (8.8 mL)swas added. After 30 min incubation, the
2
2 4
reaction was stopped by addition of 25 µL/well 0.5 M H SO ,
and optical densities were measured at 405 nm.
Virus Preparation. The pNL4-3 construct (8 µg) was
transfected into 293T cells by Lipofectamine LTX (Invitrogen,

Synthetic Antigen Inducing Structural Specific Antibody
Bioconjugate Chem., Vol. 21, No. 4, 2010 711
Scheme 1. Synthesis of the Equivalently Branched Template 6
followed by oxidative cleavage of diol group led to the desired
template 6. This approach uses thiazolidine ligation for chemose-
lective coupling of Cys-containing unprotected N36RE
(N36REGC) with a three-armed aldehyde scaffold producing
triN36e (Figure 1D). Thiazolidine ligation is a peptide segment
coupling strategy which does not require side chain protecting
groups (22-26). The reaction consists of three steps: (i) aldehyde
introduction, in which a masked glycolaldehyde ester is linked to
the carboxyl terminus of an unprotected peptide by reverse
proteolysis; (ii) ring formation, in which the unmasked aldehyde
reacts at acidic pH with the R-amino group of an N-terminal
cysteine residue of the second unprotected peptide forming a
thiazolidine ring; and (iii) rearrangement at higher pH in which
O-acyl ester linkage is converted to an N-acyl amide linkage
forming a peptide bond with a pseudoproline structure (Figure 2).
Circular dichroism (CD) spectra of triN36e and N36RE,
which is a monomer form without N-terminal Cys-Gly residues,
are shown in Figure 3A. The peptides were dissolved in 20 mM
acetate buffer with 40% MeOH, pH4.0, suitable for measure-
ment of CD spectra of membrane proteins (27, 28). Both spectra
display double minima at 208 and 222 nm and showed high
molar ellipticity as absolute values (Table 1). The results indicate
that these peptides form a highly structured R-helix and that
the helical content of the trimer triN36e is higher than that of
the monomer N36RE. Furthermore, to assess the interaction of
triN36e with C34, CD spectra of the peptide mixture with C34-
derived peptide, C34RE, were measured (Figure 3B,C). The
spectrum of triN36e and C34RE mixture showed high molecular
ellipticity as an absolute value comparable with that of triN36e
alone. This supports the conclusion that C34RE interacts with
tri36e and thereby induces a higher helical form as shown
previously (29).
antibody (2C2; 1:2000 dilution) (20) and anti- mouse IgG
(
H+L)-HRP (Millipore, MA). The band intensity of p24 was
calculated with post/pre-immunized samples by using ImageJ
Mice were immunized with these synthetic gp41 mimetics
and antibody production was successfully induced (the detailed
titer increase in 5 weeks’ immunization is given in the
Supporting Information). Two out of three mice showed
induction of antibodies against either antigen (N36RE or
triN36e). Antibody titers and selectivity of antisera isolated from
mice immunized with N36RE or triN36e were evaluated by
serum titer ELISA against coated synthetic antigens. The most
active antiserum for each antigen was utilized for the evaluation
of binding activity by ELISA (Figure 4). The N36RE-induced
antibody showed approximately 5 times higher affinity for
N36RE than for triN36e, as 50% bound serum dilutions are
image analyzing software.
RESULTS AND DISCUSSION
The N-region of gp41 is known to be an aggregation site
involving a trimeric coiled-coil conformation. In design of an
N36-derived peptide (N36RE), the triplet repeat of arginine and
glutamic acid was fused to the N-terminus to increase the
solubility in buffer solution (Figure 1B). In order to form a triple
helix corresponding precisely to the gp41 prefusion form, we
designed the novel C3-symmetric template depicted in Figure
1
C. This designed template linker has three branches of equal
-
4
-3
length and possesses the hydrophilic structure and ligation site
for coupling with N36RE. The template was synthesized from
the commercially available 3-[bis(2-carboxyethyl)amino]pro-
panoic acid 1 as shown in Scheme 1. Coupling of 1 with
3.88 × 10 and 2.14 × 10 to N36RE and triN36e,
respectively. It is noteworthy that the triN36e-induced antibody
showed approximately 30 times higher preference in binding
affinity for triN36e antigen than for N36RE (serum dilutions at
-
3
-4
ꢀ
-alanine benzyl ester 2 gave the corresponding triamide 3 in
7% yield. Cleavage of three benzyl esters by hydrogenation
and coupling with solketal 4 produced the corresponding triester
. Deprotection of the acetonides with aqueous 80% TFA
50% bound are 3.83 × 10 to N36RE and 1.33 × 10 to
triN36e). Although this evaluation was not determined with
purified mAbs, it is clear that the antibodies produced exploit a
structural preference for antigens. The mechanism of induction
7
5
Figure 2. Reaction mechanisms of thiazolidine ligation utilized for assembly of N36RE helices on the template.

712 Bioconjugate Chem., Vol. 21, No. 4, 2010
Nakahara et al.
Figure 3. (A) Circular dichroism (CD) spectra of N36RE and triN36e. In the spectra, a blue dashed line and a green line show N36RE (monomer)
and triN36e (trimer), respectively. Concentrations of the peptides are 10 and 3.3 µM for N36RE and triN36e, respectively. (B) CD spectra in the
presence or absence of C34RE peptide. The spectra show the following: a dashed green line, triN36e; a dashed blue line, C34RE; a red line,
triN36e+C34RE, respectively. The concentrations of peptides were as follows: triN36e (2.3 µM), C34-derived peptide C34RE (7 µM), and mixture
of both peptides (3.5 µM each). (C) The amino acid sequence of C34RE described in single letters. FP and TM represent hydrophobic fusion
peptide and transmembrane domain, respectively.
Figure 4. Serum titers of antibodies produced by N36 monomer and conformationally constrained N36 trimeric antigen. The titers were evaluated
against N36RE (monomer) (A) and triN36e (trimer) (B). The plots indicate the results of sera obtained from N36RE-immunized mouse (•) and
triN36e-immunized mouse (9).
Figure 5. Determination of neutralization activity of the antibodies produced by immunization of peptidomimetic antigens. (A) Results of p24
assay to evaluate inhibition for HIV-1 infection by produced antibodies. Preimmunization sera were used as control. Experiments were duplicated.
(
B) Average % inhibition of p24 production calculated from the band intensities in panel (A).
of structure-specific antibody is still not clear, but the results
could suggest the efficacy of producing antibodies with structural
specificity and that the synthesis of structure-involving antigens
is an effective strategy when higher specificity is required.
Neutralizing activity of sera against HIV-1 infection was
assessed by p24 assays utilizing antisera from two mice that
showed antibody production for each antigen (Figure 5). Sera
Table 1. Differences of r-Helicities between N36RE and triN36e
3
Calculated from CD Spectra in Figure
[θ]222
[θ]222/[θ]208
R-helicity
N36RE
triN36e
-30 957
-38 998
0.87
0.96
73%
95%

Synthetic Antigen Inducing Structural Specific Antibody
Bioconjugate Chem., Vol. 21, No. 4, 2010 713
Table 2. EC50 and CC50 Values Calculated from Inhibition Assays
of Peptidomimetics
ACKNOWLEDGMENT
The authors deeply thank Prof. K. Akiyoshi (Tokyo Medical
and Dental Univ.) for allowing access to CD spectroporalimeter.
AZT
triN36e
N36RE
a
b
EC50 (µM)
0.047
>50
0.49
>1
1.4
>10
CC50 (µM)
Supporting Information Available: HPLC chromatograms
and NMR charts of compounds 3, 5, and 6. Results of ESI-
TOF-MS, and HPLC chromatograms of peptides N36RE,
N36REGC, and triN36e. Results of serum titer ELISA of
antisera collected during immunization. This material is available
free of charge via the Internet at http://pubs.acs.org.
a
EC50 values are based on bthe inhibition of HIV-induced
cytopathogenicity in MT-4 cells. CC50 values are based on the
reduction of the viability of MT-4 cells. All data are the mean values
for at least three experiments.
from mice immunized with the same antigen showed similar
inhibitory activity against viral infection (12.5% and 14.8% for
N36RE, 40.3% and 52.1% for triN36e). A trend was observed
that the sera from triN36e immunization shows higher inhibition
than those from N36RE immunization. This suggests that the
synthetic antigen corresponding to the N36 trimeric form induces
antibody with neutralization activity superior to that of the
monomer peptide antigen and implies a restricted response of
B-cells upon immunization to the trimeric form of N36RE. In
order to assess the compatibility of induced antibodies in HIV-1
entry inhibition, the HIV-1 inhibitory activities of peptidomi-
metics (N36RE and triN36e) have been evaluated by viral
infection and cytotoxicity assays. A C-terminal region peptide
known as Enfuvirtide (T20, Roche/Trimeris) has been used
clinically as a fusion inhibitor, and its success indicates that
gp41-derived peptides might be potent inhibitors, useful against
HIV-1 infection (30). In the development of anti-HIV peptides,
several mimetics such as Enfuvirtide, CD4 binding site of gp120
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ligation attached the esteraldehyde of three-branched template
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