From the
A series of monoclonal antibodies has been generated against an
hexapeptide of sequence IRGERA corresponding to the C-terminal residues
130-135 of histone H3 and three analogues of this model peptide.
The analogues correspond to the D-enantiomer, containing only
D-residues, and two retro-peptides containing NH-CO
bonds instead of natural amide peptide bonds. The chirality of each
residue was maintained in the retro-peptide and inverted in the
retro-inverso-peptide. Monoclonal antibodies were generated from mice
immunized with the analogues coupled to neutral small unilamellar
liposomes containing monophosphoryl lipid A as adjuvant. The reactivity
of antibodies with the four analogues and with the parent protein H3
was studied in enzyme-linked immunosorbent assay and in a biosensor
system. The equilibrium affinity constant
(K
Studies of the relationship between peptide conformation and
activity have shown that the introduction of backbone modifications
into biologically active peptides affects potency, enzymatic stability,
and conformation properties, which, in some cases, leads to the
production of analogues with advantageous properties
(1) . For
instance, pseudopeptides containing reduced peptide bonds
In view of recent progress toward the preparation of pseudopeptides
and peptide mimetics for obtaining useful drugs structurally related to
their parent peptides, the design of such structures to replace natural
peptides in immunology may be promising. However, in contrast with
intense research developed in the domain of peptide drug design, very
few studies have been performed with peptidomimetics in immunology.
Potential applications of pseudopeptides and peptidomimetics cover many
aspects of basic immunology including synthetic vaccines,
immunodiagnostics, and the development of new generations of
immunomodulators. Recently, we have analyzed the antigenic and
immunogenic properties of an all D-, a retro- and a
retro-inverso-analogue of the model hexapeptide of sequence IRGERA
corresponding to the COOH-terminal residues 130-135 of histone
H3
(8, 9) . Both retroanalogues contained NH-CO
bonds instead of the CO-NH peptide bonds; the chirality of each
residue was maintained in the retro-peptide, and inverted in the
retro-inverso-peptide. It was found that the three analogues induced in
mice IgG antibodies of various subclasses. While IgG3 antibodies
reacted similarly with the four analogues (L-, retro-inverso-,
D-, and retro-peptides), antibodies of the IgG1, IgG2a, and
IgG2b isotypes showed a strong conformational preference for certain
analogues. Thus, regarding IgG1, IgG2a, and IgG2b antibodies, the
retro-inverso-peptide was found to mimic the antigenic activity of the
natural L-peptide but not of the all D- and
retro-peptides. Conversely, the retro-peptide mimicked the
D-peptide but not the L- and retro-inverso-peptides.
The finding that a retro-inverso-peptide can mimic and thus replace a
natural L-peptide was confirmed by the reactivity of a
particular monoclonal antibody (mAb)
In order to assess the potential use of
retro-inverso-peptides as immunogens useful for vaccination or for
eliciting antibody probes for immunotherapy, it was necessary to
analyze in more detail the capacity of antibodies generated against
these peptide analogues to react with the parent protein and
particularly with assembled complex structures. The model peptide
studied in the present investigation is particularly interesting to
study cross-reaction of anti-peptide antibodies since the region
130-135 of H3 is accessible at the surface of chromatin core
particles, constituted by two copies of each core histone H2A, H2B, H3,
and H4 and 145 base pairs of DNA. In the present study, we describe the
properties of a set of murine mAbs against the L- and the all
D-IRGERA peptides and, for the first time, against two types
of analogue, namely the retro-inverso- and retro-peptides. These
antibodies were generated from mice immunized with the analogues
coupled to neutral small unilamellar vesicles containing a nontoxic
derivative of lipid A, monophophoryl lipid A (MPLA), as adjuvant. We
have measured the equilibrium affinity constants of these antibodies in
the BIAcore toward the four analogues, the parent protein H3, and core
particles. The results present the first unequivocal example for the
potential application of retro-inverso peptidomimetics in the
generation of strongly cross-reactive antibodies.
The capacity of mAbs to recognize the four
peptide analogues and H3 was measured in the BIAcore using antigens
covalently linked to the dextran matrix through the free SH group
introduced in peptides for this purpose or through amino groups of
histone H3. Several concentrations of each mAb were allowed to react
with the various immobilized antigens. Kinetic rate constants and
equilibrium affinity constants of mAbs for the four peptide analogues
and H3 are shown in I. As described above for ELISA, mAbs
to the L- and retro-inverso-peptides bound preferentially the
L- and retro-inverso-peptides in the BIAcore system. The only
striking exceptions concerned mAb 13x13 which, as in ELISA
(), did not bind the parent L-peptide and mAb
4x8, which, in addition to binding the L- and
retro-inverso-peptide, also recognized the D- and
retro-peptide. The latter cross-reaction was not seen in ELISA
(). Interestingly, mAbs 4x8 and 4x11 (anti-L)
both had a higher equilibrium affinity value K
The
seven anti-L- and retro-inverso-peptide mAbs reacted with H3.
Depending on the mAbs, equilibrium affinity values
K
The most important finding in this work was to show that three of
the four mAbs to the retro-inverso IRGERA peptide bound equally well
the retro-inverso and the natural peptide (with
K
In terms
of structural and functional consequences in antibody-antigen reaction
of specific modifications of peptidic bonds in a peptide, the results
indicate that in our system, antibody specificity is not affected when
the backbone of the IRGERA peptide is changed. In contrast, we show
evidence that antibody interaction does not occur when, instead of
presenting naturally-oriented side chains, the IRGERA peptide contains
side chains with the same charge but a mirror orientation. The
mechanism of IgG3 antibody interaction is apparently different. The
fact that IgG3 Mabs induced against the D-enantiomer react
with the L-peptide and H3 and, conversely, that IgG3 Mabs
induced against the L-peptide react with the all
D-peptide
(8, 9) supposes that for this class
of antibodies, the orientation of side chains is not important.
Understanding the details of fine mechanisms of this interaction should
be gained by further structural and dynamic studies by x-ray
crystallography and NMR.
In the context of vaccine design, the
demonstration that antibodies to a retro-inverso-peptide can
cross-react particularly well with the cognate nucleoprotein structure
is very important and promising for further development. The antibodies
described in this study have been generated against the peptide
analogue covalently bound to liposome containing the nontoxic adjuvant
MPLA. Since the present study was performed with the C-terminal region
of H3, which is known to be highly accessible and probably particularly
mobile, the results should be confirmed with other exposed protein
domains, which correspond to internal sequences in the primary
structure of the protein. However, collectively, the strategy is
encouraging in respect to the potential use of retro-inverso-peptides
in the design of much more potent synthetic vaccines. We are currently
investigating the immunogenicity of retro-inverso-peptides
corresponding to neutralization epitopes, and other types of peptide
bond modification are also under investigation
(23) .
Theoretically, a vaccine consisting of such modified peptides in
combination with T-epitopes associated with immunosomes may be
effective in producing neutralizing antibodies against complex
infectious structures.
Microtiter
plates were coated with 2 µM peptide conjugated to bovine serum
albumin (BSA) (carrier to peptide molar ratio 1:10) or with 100 ng/ml
H3 and allowed to react with 4 µg/ml mAb. Anti-mouse IgG-peroxidase
conjugate was diluted 1:5000. Results are expressed in terms of:
-, absorbance values measured at 450 nm <0.4; +,
0.4-1.0; ++, >1.0->2.0; +++,
>2.0->3.0. L, natural peptide; RI, retro-inverso-peptide;
D, D-peptide; R, retro-peptide.
NB, no binding. Association (k
For details, see the legend of Table III.
We thank Dr. M. Monestier for the gift of mAb
LG11-2, Dr. M. Friede for the preparation of liposomes, and G.
Sommermeyer for skilled technical assistance in the preparation of
mAbs.
) toward the retro-inverso-peptide of
two out of three antibodies of IgG1 isotype induced against the
L-hexapeptide was 7-75-fold higher than toward the
homologous L-peptide. The range of K
values of four antibodies of IgG1 and IgG2a isotypes generated
against the retro-inverso-peptide was 0.6-1.9
10
M
for both the retro-inverso- and
L-peptides. Furthermore, antibodies to the L- and
retro-inverso-peptides cross-reacted strongly (in some cases better
than with the homologous peptide) with the parent histone H3 and with
chromatin subunits containing H3. The results are thus promising in
respect to the potential use of retro-inverso-analogues, which are
particularly stable, in the design of much more potent synthetic
vaccines or to generate antibody probes.
-(CH
-NH) or retro-inverso modifications
-(NH-CO) have been particularly investigated both in
biological and structural
studies
(2, 3, 4, 5, 6, 7) .
(
)
induced
against the L-peptide (mAb 4x11), which recognized the
retro-inverso-peptide (equilibrium affinity constant
K
, 2
10
M
) much better than the parent
L-peptide (K
, 3
10
M
). Furthermore, we have also shown
that the retro-inverso-peptide was much more resistant to proteolysis
than the L-peptide. Its half-life in the presence of trypsin
was at least 7 times longer.
Histone H3, Chromatin Core Particles, and
Peptides
Core particles and histone H3 were isolated from
chicken erythrocytes and purified as described previously
(10) .
The four peptide analogues were synthesized by the solid phase
methodology on a multichannel peptide synthesizer
(11) . For
retro-inverso- and retro-peptides, the end group-modified retro- and
retro-inverso-isomers were assembled in t-butyloxycarbonyl
chemistry on a p-methyl benzhydrylamine resin (Applied
Biosystem, Roissy, France). Protected amino acids were from Neosystem
(Strasbourg, France). Assembly of the protected peptide chain was
carried out on a 200-µmol scale using the in situ neutralization protocol described previously
(11) . The
(R,S)-2-methyl malonic acid monobenzyl ester obtained
by alcoholysis of 2,2,5,-trimetyl-1,3-dioxane-4-6-dione
(12) was incorporated into the peptide chain as a racemate. The
coupling was monitored with the ninhidrin test. After this last
coupling, the peptide resin was washed twice with ether and dried under
vacuum in a dessicator. The peptides were cleaved from the resin by
treatment with anhydrous HF containing 10% (v/v) anisol and 1% (v/v)
1,2-ethanedithiol. After removal of HF in vacuo, the peptides
were extracted from the resin and lyophilized. The crude peptides were
then purified on a C18 column using a middle pressure apparatus
(Kronwald Separation Technology, Sinsheim, Germany) by elution with a
linear gradient of 5-50% (v/v) acetonitrile in aqueous 0.06%
trifluoroacetic acid. The purity of each fraction was assessed by
analytical runs on a Novapack C18 column 5 µm (3.9 150 mm)
using a linear gradient of 7-32% (v/v) acetonitrile in aqueous
0.1 M triethylammonium phosphate buffer. Runs were performed
with a Waters apparatus (Waters Corp., Milford, MA). The fractions
containing the pure diastereomeric mixture were pooled and lyophilized.
Mass spectra were obtained on a VG analytical ZAB-2SE double focusing
instrument and recorded on a VG 11-250 data system (VG
Analytical, Manchester, United Kingdom) as described
previously
(13) .
Peptide-Carrier Conjugation
To allow the coating
of peptides in a direct solid phase ELISA test, IRGERA analogues were
conjugated to bovine serum albumin using N-succinimidyl
3-[2-pyridyldithio]propionate (SPDP) as described
previously
(14) . For immunization of mice, peptides were
covalently coupled to preformed small unilamellar vesicles containing
MPLA
(8, 15) .
Generation of mAbs
For mAb production, BALB/c mice
were injected intraperitoneally with the four small unilamellar
vesicles preparations containing 1 µmol of lipid, 2 µg of MPLA,
and 100 µg of peptide/injection/animal. Mice received three
injections at intervals of 3 weeks. Booster injections were given at
days 105, 106, 107, and 108, i.e. -4, -3,
-2, and -1 before fusion. mAbs were prepared by standard
fusion protocols
(16) . Spleen lymphocytes were mixed at a 1:1
ratio with nonsecreting PAI myeloma cells
(17) . The cells were
distributed in 96-well plates at a concentration of 2 10
cells/ml. Starting 7 days after the fusion, culture supernatants
were tested by ELISA for the presence of specific antibodies reacting
with peptide analogues and H3. Positive cultures were recloned twice in
agar in the presence of 10
macrophages/ml as feeder cells.
Positive clones were amplified by in vitro culture in
Dulbecco's modified Eagle's medium containing 5% fetal calf
serum and glutamax (Life Technologies, Inc.). Antibodies (in 370-1000
ml of culture medium) were subsequently precipitated with ammonium
sulfate at 50% saturation. Some of them were further purified by
protein A-Sepharose chromatography. Their purity was checked by 12%
SDS-polyacrylamide electrophoresis. The antibody concentration was
determined by optical density measurement at 280 nm using an absorption
coefficient of 1.4. Protein concentration was also determined using the
Bio-Rad protein assay (Bio-Rad).
ELISA and Kinetic Analysis of mAb Binding
The
ELISA procedures (direct binding experiments and determination of mAb
isotypes) were as described previously
(8) . For real time
binding experiments, a BIAcore biosensor system (Pharmacia Biosensor,
AB, Uppsala, Sweden) was used. Reagents including sensor chips CM5,
surfactant P20, and coupling kit containing
N-hydroxysuccinimide,
N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide (EDC),
2-(2-pyridinyldithio)ethane amine (PDEA) and ethanolamine HCl were
obtained from Pharmacia Biosensor AB. To immobilize peptides to the
sensor chip, the carboxyl-dextran matrix was first activated with 0.2
M EDC and 0.05 MN-hydroxysuccinimide. It
was further modified by injecting PDEA thiol coupling reagent (15
µl of 80 mM PDEA in 0.1 M borate buffer, pH 8.5)
allowing the thiol reactive group of peptide analogues to be coupled to
the activated matrix. After the peptide immobilization run, remaining
reactive groups on the sensor surface were deactivated by a 4-min pulse
of 50 mM cysteine in 1 M NaCl, pH 4.3. Histone H3 was
immobilized by the conventional procedure using EDC and
N-hydroxysuccinimide-activated dextran matrix. Core particles
were immobilized by trapping them on sensor chips containing covalently
bound rabbit anti-mouse IgG Fc and a purified murine mAb directed
against histone H2B (mAb LG11-2; a gift of M. Monestier,
Philadelphia, PA) as a second layer. mAbs were then allowed to interact
with sensor chips on which the four peptide analogues, H3, or core
particles had been immobilized. 6-10 concentrations of each mAb
ranging from 50 to 800 nM in HBS, pH 7.4 (10 mM
HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore
surfactant P20) were used in each test. The antibody preparation was
injected at a constant flow rate of 5 µl/min during 7 min at 25
°C, and report points for calculation were taken every 10 s during
5 min, starting 1.5 min after the end of mAb injection. Antibody
kinetic constants were measured as described previously
(18) .
Theory of kinetic measurementsusing the BIAcore biosensor system has
been described previously
(19, 20, 21) .
RESULTS
Generation of mAbs to the L-Parent Model
Peptide IRGERA and IRGERA Analogues
Four groups of two BALB/c
mice were injected with the L-, retro-inverso-, D-,
and retro-IRGERA analogues coupled to small unilamellar vesicles
containing MPLA as adjuvant, and four fusion experiments were performed
with the spleen cells of these mice. Characteristics of retro-inverso-
and retro-analogues, which contain reversed peptide bonds (i.e. NH-CO instead of CO-NH) have been described previously
(Fig. 1)
(9) . The four peptides contain a cysteine and two
additional glycine residues at the NH-terminal end to allow
selective conjugation of the peptides to bovine serum albumin and
liposomes, and to enhance their accessibility when bound to carriers.
ELISA reaction with peptide analogues and with H3 of polyclonal mouse
antisera raised against these four peptides have been described
previously
(8, 9) . Hybridomas secreting antibodies to
peptide analogues and H3 were screened by ELISA using antigens adsorbed
to plastic of microtiter plates, and positive cultures were recloned
twice in agar. A total of 67 stabilized specific hybridomas were
obtained from the four fusions (). A number of these clones
were of the IgG3 isotype. Since the reactivity of IgG3 antibodies in
regard to their recognition of peptide analogues appeared quite
different from that of antibodies of the IgG1, IgG2a, and IgG2b
subclasses
(8, 9) , data obtained with IgG3 mAbs will not
be described here. Nine positive clones secreting IgG1, IgG2a, and
IgG2b antibodies () were amplified in vitro,
purified, and tested for their ability to cross-react with the parent
IRGERA peptide, peptide analogues, the parent protein histone H3, and
chromatin subunits. Between 35 and 63 mg of each mAb were available for
these measurements.
Figure 1:
Schematic structure of the natural
L-peptide IRGERA and retro-inverso
analogue.
Reactivity in ELISA and in the BIAcore of mAbs to IRGERA
and IRGERA Analogues
The capacity of the nine mAbs to react in
ELISA with IRGERA peptides and H3 is summarized in . In
most cases, mAbs to the L- and retro-inverso-peptides
cross-reacted more or less strongly with the L- and
retro-inverso-peptides as well as with the parent protein H3, while
mAbs generated from mice immunized with the D- and
retro-peptides cross-reacted with the D- and retro-peptides
but not with the L- and retro-inverso-peptide and with H3.
These results are thus in complete agreement with those described
previously with mouse antisera raised against the four IRGERA
peptides
(9) . The only exception was found with mAb 13x13
(anti-retro-inverso) which did not react with the parent
L-peptide while, intriguingly, it recognized the whole histone
H3 ().
for the retro-inverso-analogue compared with
K
values for the homologous peptide. In
the case of mAb 4x11, this was due to a lower dissociation rate
constant. Association and dissociation rate constants of mAbs 13x12,
13x14, and 13x18 with respect to the L- and
retro-inverso-peptides were very similar for each mAb. mAbs 11x4
(anti-D-peptide) and 12x10 (anti-retro-peptide) presented
similar k
and k
values with respect to the retro- and D-peptides.
were between 0.3 and 6.5 10
M
. mAbs 4x8 and 4x11
(anti-L-peptide) showed a much higher affinity for H3 than for
the parent L-peptide (54 and 76 times, respectively). As
expected, mAbs 11x4 (anti-D-peptide) and 12x10
(anti-retro-peptide) used as control antibodies did not recognize H3.
Reactivity of mAbs to IRGERA and IRGERA Analogues with H3
in Core Particles
Since several mAbs generated against IRGERA
and the retro-inverso-analogue recognized the parent histone H3, their
capacity to recognize H3 in more complex structures such as core
particles was further studied. Core particles are the basic repeating
structural subunits of eukaryotic chromatin and are composed of two
molecules of each histone H2A, H2B, H3, and H4 surrounded by 145 base
pairs of DNA. When adsorbed to a solid surface, a part of the
conformation of these subunits may be affected. Likewise, the
conformation of core particles may be considerably altered when they
are covalently bound to the dextran matrix of the sensor chip via
primary groups, particularly because histone tails, which are very
basic, play an important role in the stabilization of the edifice. To
overcome this problem, isolated core particles were captured by a first
mAb directed against histone H2B, which was presented by rabbit
anti-mouse IgG Fc immobilized on the sensor chip. Anti-H2B mouse mAb
LG11-2 was shown in independent experiments to specifically
recognize peptide 1-25 of H2B.(
)
A typical
immobilization plot of presenting antibodies (rabbit anti-mouse IgG and
mAb LG 11-2) and the binding of core particles is shown in
Fig. 2
. This figure also shows an example of interaction between
mAb 4x11 and core particles. As shown in , six of the
seven mAbs induced against the L- and retro-inverso-peptide
reacted with core particles. The equilibrium affinity constants
K
of mAbs 4x8, 4x10, and 4x11
(anti-L-peptide) for core particles were 1.2
10
M
, 5.9
10
M
, and 1.2
10
M
, respectively.
K
values of mAbs 13x12, 13x14 and 13x18
(anti-retro-inverso-peptide) for core particles were 1.7
10
M
, 1.9
10
M
, and 2.2
10
M
, respectively. mAb 13x13 did not
recognize core particles in the BIAcore system. As expected, the mAbs
induced against D- and retro-peptides did not react with core
particles.
Figure 2:
Sensorgram showing the immobilization of
chromatin core particles on the sensor chip and an example of mAb
binding. The flow rate was 5 µl/min. A, activation of the
carboxylated dextran matrix by the injection of 30 µl
EDC/N-hydroxysuccinimide mixture; B, injection of 35
µl of a 100 µg/ml solution of rabbit anti-mouse IgG Fc in HBS,
pH 7.4; C, inactivation of unreacted groups by 35 µl of
ethanolamine; D, washing off noncovalently bound rabbit
anti-mouse IgG Fc with 15 µl of 0.1 M HCl; E,
injection of 10 µl of anti-H2B mAb LG11-2 (400 nM);
F and G, injection (2) of a nonrelated ascitic
fluid diluted 1:5; H, injection of 10 µl of core particles
(200 nM) in HBS, pH 7.4; I, injection of 35 µl of
mAb 4x11 (200 nM). mAbs were allowed to react 8 min with
captured core particles at a flow rate of 5 µl/min. J,
injection of 15 µl of 0.1 M HCl. Arrows indicate
start and end of injections. Steps E-H were
repeated for each new injection of anti-peptide
mAb.
DISCUSSION
The IRGERA peptide provides an excellent model for the study
of antigenic and immunogenic properties of pseudopeptides. It is known
to be non-immunogenic in the absence of a carrier protein and
particularly sensitive to proteases, and because this region of H3 is
exposed at the surface of nucleosome in chromatin
(22) ,
anti-peptide antibodies are interesting probes to study antibody
interaction with complex nucleoprotein particles. In this study, we
have investigated the specificity and affinity of mAbs induced against
the natural L-hexapeptide and analogues containing reversed
amide bonds. The retro-inverso transformation has been used by numerous
bioorganic chemists to convert biologically active peptides into more
stable compounds
(2, 3, 6) . We have shown
previously
(9) that the retro-inverso IRGERA peptide was much
more resistant to trypsin than the corresponding natural peptide.
values ranging from 0.6 to 1.9
10
M
). The four mAbs reacted
with the cognate protein (K
values,
0.3-2
10
M
), and
three of them reacted with chromatin core particles presented toward
the liquid phase by the N-terminal end of histone H2B
(K
values, 0.2-2.2
10
M
). In contrast, mAbs induced against
the all D-peptide and the retro-analogues, used as control
antibodies in this series of experiments, did not recognize the natural
peptide, histone H3 and H3 within isolated core particle.
Table:
Summary
of results obtained in fusion experiments of splenocytes from mice
immunized against the L-parent IRGERA hexapeptide and peptide
analogues
Table:
Reactivity in
ELISA of mAbs induced against IRGERA and IRGERA analogues
Table:
Kinetic rate constants and equilibrium
affinity constants of mAbs induced against IRGERA and IRGERA analogues
) and
dissociation (k
) rate constants are the
mean values obtained in two to four independent experiments.
L, natural L-peptide; RI, retro-inverso-peptides;
D, D-peptide; R, retro-peptide. mAbs 4
8, 4
10, and 4
11 are anti-L-peptide antibodies;
mAbs 13
12, 13
13, 13
14, and 13
18 are
anti-retro-inverso-peptides; mAb 11
4 is an
anti-D-peptide and mAb 12
10 is an anti-retro-peptide.
Table:
Kinetic rate constants and equilibrium affinity
constants of mAbs induced against IRGERA and IRGERA analogues with
respect to core particles
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.