immunogenic levels. A series of D-analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3.
VOl. 268, No. 35,Issue of December 15,pp. 26279-26285,1993 Printed in U.S.A.
THEJOURNAL o? BIOLOGICAL CHEMISTRY
Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Antigenicity and Immunogenicity of Modified Synthetic Peptides Containing D-Amino AcidResidues ANTIBODIES TO A D-ENANTIOMER DO RECOGNIZE THE PARENTL-HEXAPEPTIDE AND RECIPROCALLY* (Received for publication, July 9,
1993, and in revised form, August 10,
1993)
Nadia Benkirane, Martin Friedej,Gilles Guichard, Jean-Paul Briand,Marc H. V. Van Regenmortel, and SylvianeMuller$ From the Znstitut de Bwbgie Mokculaire et Cellulaire, UPR 9021 Centre National de la Recherche Scientifique,15 rue Descartes, 67000 Strasbourg, and the §Laboratoire de Chimie Bioorganique, UR.A 1386 Centre National de la Recherche Scientifique, Fmulte de Phurmacie, 74 Route du Rhin, 67400 Zllkirch, France
The effect of introducing D-amino acid residues in Over the last decade, several candidate synthetic vaccines an hexapeptide was examined both at theantigenic and have been tested up to the level of in vivo protection trials. immunogenic levels. A series of D-analogues of the There has been an increasing tendency to design peptides model peptide of sequence IRGERA corresponding to endowed with a particular conformation and several peptides the COOH-terminal residues 130-135 of histone H3 have been assessed as potentialvaccines (Brown, 1990; Milich, were produced. Four analogues contained a single 1990; Arnon and Van Regenmortel, 1992). Of particular inchange of an L-residue by the corresponding enan- terest is the possibility of improving the antibody response, tiomer, one peptide contained two D-residues and an- in terms of specificity, isotype, and duration, by altering the other one contained only D-residues @-enantiomer). A presentation or composition of a peptide. To thisend, peptide peptide analogue was also synthesized in which the 2 analogues containing D-amino acid residues or modified pepArg residues were replaced by Lys residues. The parent peptide and peptide analogues were injected into tide bonds which exhibit a much higher resistance to proteases mice after covalent coupling to small unilamellar li- may be rendered, by virtue of their increased stability, more posomes containing monophosphoryl lipid A as adju- immunogenic than the native peptide. Such modifications vant. The substitution of ~-Arg"l to Lys or D-Arg was have found many applications in the field of peptide-based found to change neither the antigenic norimmunogenic drugs where increased stability has resulted in significantly properties of the resulting peptides. In contrast, the increased biological activities (Fauchgre and Thurieau, 1992). substitution of Glul", Arg"', and Ala"' by the respec- In contrast to studiesdealing with pharmacological activities tive enantiomers drastically altered the antigenicity of of peptides containing D-residues, very little has been pubthe modifiedpeptides. Each of the six analogues lished regarding the antigenic and immunogenic properties of induced an immune response with an unusually high such analogues (Geysen et al., 1986, 1987). In the present level of IgG3 antibodies. The D-enantiomer produced study, we have evaluated the influence of L- to D-amino acid IgG3 antibodies which reacted with the homologous substitutions on the antigenic and immunogenic properties of peptide as well as with the all L-peptide and the parent a model hexapeptide. A series of peptide D-analogues (several protein H3 in solution but not with analogues contain- partially modified analogues and the mirror D-enantiomer; ing one or two D-residues only. IgG3 antibodies pro- Wade et al., 1990) were testedin the hope of defining if duced against the all L-peptide reacted with the free changes in peptide structure lead to a longer duration of the all D-peptide but not with the other analogues contain- immune response without impairing the capacity of modified ing D-residues in position 133, 134, and135. peptides to induce antibodies cross-reacting with the cognate protein. In order to minimize any influence of the carrier, the parent peptide and analogues were injected into mice after Synthetic peptides have received considerable attention as coupling to small unilamellar liposomes containing monopotential vaccines because they are chemically defined, have phosphoryl lipid A as adjuvant. The short peptide IRGERA very long shelf lives, and are likely to be safer and cheaper which alone is not immunogenic (Frisch et al., 1991)was than conventional vaccines derived from infectious material. selected for this study. A short peptide was used since it The design of a synthetic vaccine requires a thorough knowl- seemed likely that with longer peptides, the presence of Dedge of the mechanisms of protective immunity involving B amino acids may also affect processing and presentation to and T epitopes and of the parameters that affect peptide MHC molecules (Milton et al., 1992; Jung, 1992) in addition to altering peptide stability. immunogenicity such as the mode of presentation to the immune system, the need for a carrier and adjuvant, and the MATERIALS AND METHODS influence of route and method of delivery. *This work was supported by Centre National de la Recherche Scientifique GCCB Project 28D4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 To whom correspondence should be addressed Institut de Biologie Molkulaire et Cellulaire, Laboratoire d'Immunochimie, 15 rue Descartes, 67084 Strasbourg Cedex, France.
Peptides-Assembly of the protected peptide chains was carried out on a multichannel peptide synthesizer (Neimark and Briand, 1993)using the procedure described previously (Friede et al., 1992). Briefly, all amino acids were protected a t the a-amino position with the Fmoc group. The following side chain protecting groups were used tert-butyl ester for Glu and 2,2,5,7,8-pentamethyl-chroman-6sulfonyl for Arg. Twenty-five pmolFmoc-Ala or Frnoc-D-Ala 4hydroxymethyl phenylmethyl polystyrene resin (Novabiochem, Switzerland) were placed in each reaction vessel. Fmoc amino acid deriv-
26279
26280
D-Analogues Peptides of Antigenic
atives were coupled in 500 pl of dimethylformamide by using a BOP/ working dilution 1:40,000) or with rabbit anti-mouse IgG (H+L) HOBt coupling procedure (Hudson, 1988).Double couplings were conjugated to peroxidase (Nordic, Tilburg, The Netherlands; working performed systematically. At the end of the synthesis and after the dilution 1:5,000). After 30 min of incubation and washing with PBSlast deprotection step, the peptide resin was washed with dichloro- T, the final reaction was visualized by incubation with 3,3',5,5'methane and dried under nitrogen. The trifluoroacetic acid cleavage tetramethylbenzidine in the presence of HzOz (Briand et aL, 1992). was performed on the machine in the same reaction vessels by using The resulting absorbance was measured at 450 nm. King's reagent (King et al., 1990). Four ml of this reagent were To determine antibody isotypes, the unlabeled rabbit anti-mouse delivered into each reaction vessel. The total cleavage time was set specific for IgG1, IgGZa, IgGZb, IgG3, IgA, and IgM from Nordic were to 2 h 30 min. At the end each peptide was collected in a polypropylene used (working dilutions 1:5,000). After 30 min of incubation at 37 'C tube containing 25 ml of cold ether, centrifuged, washed again with and washings, goat anti-rabbit IgG conjugate diluted 1:30,000was ether, solubilized in 5 mlof H20/CH3CN (v/v), and lyophilized. added for 30 min at 37 "C. The other steps of the assay were as Homogeneity of the crude peptides was assessed by analytical runs described above. A number of experiments were also performed using on a Novapak Cl8 column, 5 pm (3.9 X 150 mm) using a triethyl- goat anti-mouse IgG3 conjugated to horseradish peroxidase (Nordic, ammonium phosphate buffer system. Linear gradients detected a t working dilution 1:7,000). 210 nm werefrom 1-21% CH3CN in 12 mn, and theflow rate was 1.4 For competition experiments, various concentrations of the pepml/min. Runs were performed with a Watersapparatus (Waters tides or of H3 used as inhibitors were incubated for 1 h at 37 'C and Corporation, Milford, MA). then overnight at 4 "C with antisera diluted in PBS-T. The mixture CD-Circular dichroism measurements were performed on a Jobin was then added to protein or peptide-coated wells, and the test was Yvon Dicrograph I11 under constant nitrogen flush a t 25'C. The performed as described above. In order to provide a semiquantitative spectra of peptides were recorded between 195 and 260 nm in triflu- comparison of the relative reactivities of antibodies for the peptides oroethanol (TFE)' using a 1-mm path length quartz cell. The results and H3in inhibition studies, we made the following assumptions. In were expressed as mean residue ellipticity values (e) having the units the case of BSA-conjugated peptides, we assumed that the maximal deg .cm2.dmol". binding of BSA coated into the wells of PVC microtiter plates was Peptide Carrier Conjugation-Three methods were used to conju- 400 ng (Sorensen and Brodbeck, 1986; Butler etal., 1992),correspondgate peptides to ovalbumin or bovine serum albumin (BSA). Coupling ing to 0.062 nmol of peptide. In the case of individual histones, we was performed using either glutaraldehyde, N-succinimidyl3-[2-pyr- showed previously using radioactive molecules that in the coating idyl dithiolpropionate (SPDP), or photochemical activation involving conditions used, nearly 90% of the antigen introduced in wells bethe benzophenone species benzoylbenzoyl glycine which was added comes attached to the plastic (Muller and Van Regenmortel, 1989). to the NH2-terminal residue of the peptide IRGERA at the end of This corresponds to about 20 ng H3/well. In the case of both BSA the synthesis (Muller, 1988). The yield of coupling was obtained by conjugates and histone H3, we assumed that 100% of the bound determining the amino acid composition of the final conjugate antigen was available for binding to the antibodies present in the (Briand et d.,1985) or by determining spectrophotometrically the liquid phase. release of 2-thiopyridone from SPDP derivatized BSA on interaction with cysteine-containing peptides (Muller, 1988). RESULTS Preparation of Liposome-associatedPeptides-Peptides were covaSynthetic Peptides-Eleven peptideswere used inthis study lently coupled to preformed small unilamellar vesicles (SUV) containing 4-(p-maleimidophenyl)butyrylphosphatidylethanolamine (Table I). Peptides 1-4 correspond to the natural sequence of (MBP-PE) (Friede et al., 1993). For immunization, monophosphoryl the COOH-terminal end of histone H3 (IRGERA). Th'1s relipid A (MPLA) was incorporated in SUV as adjuvant. Briefly, gion contains a major epitope of H3 which has been extenpreparation of liposome-associatedpeptideswas performed as follows: sively studied in this laboratory (Mulleret al., 1982; Frisch et SUV were formed by drying a solution of lipids (egg-yolk phosphatidyLcholine,~ cholesterol, and MBP-PE, (855015 mol %) containing al., 1991; Briand et al., 1992; Friede et al., 1993 ). In order to MPLA (Ribi, Hamilton, MO) a t 2 mg/mmol lipid, resuspension (12 enhance the accessibility of the residues in the IRGERA and pmol lipid/ml) in 100 mM NaCI, 50 mM HEPES, pH 6.5, and soni- GERA peptides bound to a carrier, two additional residues cation with a probe sonicator for 1 h. After centrifugation a t 10, 000 of Gly were added to their NH2-terminalend (peptides 1 and X g for 10 min to remove large vesicles and titaniumparticles, peptide 4-11). A Cys residue was addedat the NHz terminus of these was added at an equimolar ratio to the MBP-PE and incubated at peptides to allow selective conjugation of the peptides to BSA room temperature for 1 h. Unbound peptide was removed by dialysis. groups present on Antisera-The following rabbit antisera were used: antiserum Tri and liposomes via reaction with maleimide directed to complete histone H3, antiserum Me1 directed to peptide the activated carriers. In peptide 5, Arg131 and Arg"were IRGERA conjugated to ovalbumin by means of glutaraldehyde, and replaced by Lys residues and in peptides 6-11, D-amino acid antiserum Giz directed to peptide (benzoylbenzoylglycine) IRGERA residues replaced certain L-amino acid residues as indicated conjugated to ovalbumin by photochemical coupling. Their produc- in Table I. The replacementbyaD-opticalisomer is not tion and characterization have been described (Briand et al., 1992). applicable to the symmetrical Gly13'. The degree of purity of Mouse antiserum was obtained from BALB/c mice immunized with liposome-associated peptides. Micewere injected intraperitonealy peptides, as assessed by high performance liquid chromatogwith the various SUV preparations containing 1 pmol of lipid, 2 pg raphy, wasat least 95%.In CD measurements alonger peptide of MPLA, and 60 pg peptide/injection/animal. Mice received three (residues 118-135)was introduced as control. injections at intervals of 3 weeks. Blood was withdrawn from the Structure of Peptide IRGERA and Peptide Analogues-In retroorbital venous plexus 5 days after each injection andthen absence of structuraldata, attempts to compare the structures regularly over 6 months after the last injection. Control serum was obtained from each mouse before the firstinjection. TABLEI ELISA-The ELISA procedure used to measure the binding of Sequences of the COOH-terminalpeptides of antibodies from rabbit or mouse sera was as follows: microtiter plates histone H3 and analopues (Falcon) were coated overnight a t 37 "C with 100 ng/ml histone H3 or with 2 p~ of the various peptides and peptide conjugates in solution Sequence -__ in 0.05 M carbonate buffer, pH 9.6. After three washings of microtiter E R A'36 G GI G plates with phosphate-buffered saline containing 0.05% Tween (PBSI R G R A E T), antiserum diluted in PBS-T containing 10mg/ml BSA wasadded R A G I R E for 1 h at 37 "C. After repeated washings positive reactions were R A I R E G detected with AffiniPure goat anti-rabbit IgG (H+L) conjugated to K A E I K G horseradish peroxidase (Jackson Laboratories, BarHarbor, ME; R A I @ G E R A DE G I R ' The abbreviations used are: TFE, trifluoroethanol; BSA, bovine I R G A DR E serum albumin; SPDP, N-succinimidyl 3-[2-pyridyl dithiolpropioG R E I R E nate; SUV, small unilamellar vesicles; MBP-PE, 4(p-maleimidoDR A G E pheny1)butyrylphosphatidylethanolamine; ELISA, enzyme-linked DR G DE immunosorbent assay; MPLA, monophosphoryl lipid A. ~
D-Analogues of Antigenic Peptides
200 220
240 260
Wavelength (nm)
FIG. 1. CD spectra of peptide 118-135, peptide 4, and Danalogues in 100% TFE. Molar concentrations of peptides were 1.32 10" M for peptide 118-135 and 2.38, 2.43, 2.23, 2.50, and 2.35 10" M for peptides 4, 7, 9, 10,and 11, respectively. adopted by the different peptide analogues by molecular modeling using calculations of energy minimization were unsuccessful. No preferential conformation was observed and thus modeled structures wereof very low reliability (datanot shown)? The crystal structure of the core histone octamer has been determined at 3.1-A resolution (Arents et al., 1991). In thethree-dimensional structure the residues in region 120132 of H3 form an a-helix, while the 3 residues 133-135 are outside the helix? CD spectra show that in 100% TFE, the percent helix content inpeptide 118-135 may beestimated as 30% (Fig. 1). Helical conformation is no longer observed in peptide 4 and a negative ellipticity at 198 nm is found indicating an unordered form. Although CD spectra of peptides in TFE need to be interpreted with great circumspection, it is interesting to observe that spectra obtained with peptides 4 and 11are roughly symmetrical. Recognition in ELISA of IRGERA Analogues by Rabbit and Mouse Antibodies Induced against the Parent Peptide IRGERA and H3"Three antisera raised against the peptide IRGERA (rabbits Me1 and Giz) and histone H3 (rabbit Tri) were first tested for their ability to react with the various peptides shown in Table I. These tests were performed in a competitive ELISA using H3 as antigen for coating plates and the different peptides as inhibitors. As shown in Fig. 2 and Table 11, the binding of anti-H3 antibodies to H3was strongly inhibited by peptides 4, 5, and 6, less wellby the shorter peptide 1and not at all by peptides 7-11. Very little difference was seen when rabbit antibodies against peptide IRGERA instead of rabbit antibodies against the cognate protein H3 were used except for peptide 8 which inhibited slightly the interaction of anti-IRGERA antibodies with H3. Antisera against peptide 4 were also raised in BALB/c mice by a series of intraperitoneal injections of the peptide coupled at thesurface of SUV containing MPLA as adjuvant (Friede et al., 1993). When the antiserawere tested inthe competitive assay using H3as antigen andthe different peptides as inhibitors, very similar results to those described above with rabbitantisera were found (Table 11). Antibodies raised against peptide 4 reacted also strongly with H3 in solution (see below). The cross-reaction of antibodies against peptide 4 with the various peptide analogues and with H3 was also studied in a direct ELISA format in which H3 and the different peptide analogues conjugated to BSA by means of SPDP were used
* P. Orlewski and M-T. Cung, Nancy, France.
'E. M. Moudrianakis, personal communication.
26281
for coating plates. The conditions of coupling werecontrolled in such a way that all BSA-peptide conjugates had the same peptide to carrier molar ratio of 10. As shown in Table 111, mouse antibodies against peptide 4 reacted well with homologous peptide 4 (see also Fig. 3A) as well as with the peptide analogues 5 and 6. They also reacted strongly with the parent protein H3 (Fig. 31). In contrastthey did not bind to peptides 7-11. From the results obtained with rabbit and mouse antisera, it can be concluded that the replacement of Arg13' and Arg'34 by Lys residues (peptide 5) had not effect on antibody recognition. Likewise, the replacement in peptide 6 of Argx3'by DArg had no detectable effect on the binding of anti-H3 and anti-IRGERA antibodies. In contrast,the replacement in peptide 4 of L-residues 133,134, and 135 by the respective Disomers (peptides 7-9) dramatically altered the recognition of the peptides by anti-H3 and anti-IRGERA antibodies. Likewise, peptides 10 and 11 containing 2 D-Arg residuesin position 131 and 134 and 5 D-residues in position 130-135, respectively, were recognized neither by H3 nor by IRGERA antibodies. Mouse Antibodies to IRGERA Analogues-Groups of four BALB/c mice were injected with the various peptide analogues conjugated to liposomes. AnIgM response could be generally demonstrated by ELISA in bleeding 1which slowly decreased from bleeding 3 onward and was no longer detectable after bleeding 5 (data not shown). IgG responses to the different peptides conjugated to BSA by means of SPDP are shown in Fig. 3 (open squares). IgG antibodies were generally detected from bleeding 2 onward and, depending on the peptide used as immunogen, their activity decreased from bleeding 5 (47 days after the last injection) to 7 (89 days after the last injection). A fairly low response was found in mice immunized with peptides 7 and 11 (Fig. 3, D and H). The antibody response measured by ELISA was very similar in the individual animals of each group. Ability of Mouse AntibodiesInduced against IRGERA Analoguesto Cross-react with Histone H3 and Various Peptide Analogues-The capacity of mouse antibodies induced with IRGERA analogues coupled to liposomes to recognize histone H3, parent peptide 4, andother IRGERAanalogues was studied in different ELISA formats. The reactivity of mouse antisera was first studied in a direct ELISA in which the different antigens were adsorbed on microtiter plates (Table 111, Fig. 3, open squares). The reactivity of antibodies against peptide analogues containing Lys residues at positions 131 and 134 (peptide 5) or a D-Arg residue at position 131 (peptide 6) wasvery similar to that of antibodies against peptide IRGERA (peptide 4). These antibodies reacted with peptides 1, 4, 5, and 6 and with the cognate protein H3 but not with peptides 7-11. In contrast, antibodies induced against peptides 7-11 recognized onlythe respective homologouspeptides and did not react with heterologous peptides nor with H3. The reactivity of the mouse antibodies was also tested in a competitive binding assay with free peptides in solution to determine if the free peptides were recognized as well as the peptide conjugates. The results presented in Table IV show the molar excesses of the various peptides required to inhibit 50% of the binding between anti-peptide antibodies and the respective homologous peptides. In agreement with the results obtained with immobilized conjugated peptides, peptides 4,5, and 6 strongly inhibited the binding of antibodies against these peptides, but not that of antibodies against peptides 711. As also observed in direct ELISA, only the homologous peptides inhibited the reaction between antibodies to peptides 7-11 and their respective peptides. H3 in solution inhibited
26282
D-Analogues of Antigenic Peptides 100-
FIG. 2. Inhibition of the ELISA reaction between H3 (100 ndml) -. . and three rabbit antisera, respectively, directed to H3 (rabbit Tri, A ) , peptide 2 conjugated to ovalbumin by means of glutaraldehyde (rabbit Mel, B ) , and peptide 3 conjugated to ovalbumin by photochemical coupling (rabbit Giz, C) by increasing concentrations of peptides 1 and 4-11. A control peptide corresponding to thesequence 149-158 of tobaccomosaic virus protein (TMV-p) was usedas control. Rabbit antisera were diluted 1:3,000 and anti-rabbit IgG conjugate 1:40,000.Absorbance values were measured a t 450 nm.
A
-
50
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TABLE I1 TABLE I11 Recognition in competitive ELISA of IRGERA and IRGERA Reactivity in ELISA of mouse antibodies induced against IRGERA analogues byanti-peptide and anti-H3 antibodies and IRGERA analogues Microtiter plates were coated with 100 ng/ml H 3 and allowed to Microtiter plateswere coated with 2 F M peptide conjugated to BSA react with rabbit and mouse antisera diluted 1:3,000 and 1:500, (carrier to peptide molar ratio 1:lO)or with 100 ng/ml H 3 and allowed respectively, and preincubated with the various peptides used as to react with mouse antisera diluted 1500 (bleeding of immunized inhibitors. The reaction was revealed with anti-rabbit IgG (H+L) mice after three injections except for mice immunized with peptides conjugate and anti-mouse IgG (H+L) conjugate diluted 1:40,000and 1, 7, and 11 for which the respective bleedings 2 were used). Anti 1:5,000,respectively. Molar excesses were calculated as described mouse IgG-peroxidase conjugate was diluted 1:5,000. under “Materials and Methods.” Reactivity in ELISA with BSA-conjugated Peptides used inhibitor
Molar excess of inhibitor peptide over H3 required to obtain 50%inhibition of antibody binding to H3 with antiserum against
H3 (rabbit Tri) (rabbit
30,000 12,000 10,000 2,000 4 40 5 200 6 20 200 7 8 9 10 11
Peptide 2 Mel)
20 5 150 80,000 -
Peptide 3 (rabbit Giz)
20 4
10,000 -
Peptide 4 (mice)
200 250 300 -
’-, no detectable cross-reactivity (up to125 @g/mlpeptide).
Mouse antibodies to peptides
peptides
1 4 5 6
5
H3 protein
7
8
9
1011
+ ++ + + + + + ++ + +++++ + + + + + +++-
-
-
- - - - -
1
4
6
++a++
++ +++ +++ +++
+++ - +
10 - - - - - - 11 Results are expressed in terms of: -, absorbance values measured >1.0-1.5; +++, B1.5. The results a t 450 nm < 0.30;+, 0.3-1.0; represent the average absorbance values obtained in each group of mice (n = 4). ”
_
-
”
++,
the binding of antibodies induced against peptides 1,4,5,and 4,5,and 6 was strongly inhibited by the homologous peptides 6, but not that of antibodies against peptides 7-11, to their but not by peptide 1 and peptides 7-11 (data not shown). The respectivehomologous peptides coated as BSA-conjugates binding of H3 by antibodies to peptide 1 was only inhibited (Table V). by peptide 1. Overall, when the results obtained in the direct and comAnalysis of the Isotypesof Antibodies Raised to IRGERA and petitive ELISA tests were compared, the only difference which IRGERA Analogues Associated to SUV Containing MPLAwasobservedconcerned peptide 1. In solution thisshort The antibodies raised in BALB/c miceto the various peptides peptide was only capable of inhibiting the binding between covalently attached to SUV were essentially of the IgG type. anti-peptide 1 antibodies and peptide 1 and was unable to Antibodies of IgM isotype which were initially present gradcompetewithIRGERAanalogues (Table IV). Ina direct ually disappeared during the course of immunization, and no binding assay, this peptide coated as a BSA-conjugate was IgA antibodies were detected. Antibodies of all IgG subclasses recognized by antibodies induced against peptides 4,5,and 6 were present in the various antipeptide sera except inantisera while anti-peptide 1 antibodies recognizedconjugated pep- to peptide 11 which contained mainly IgG3 antibodies (Fig. 3H).The IgG3 subclass (Fig.3, closed squares) was also tides 4,5, and 6 (Table 111). The binding of H3 by antibodies induced against peptides prevalent in antisera to peptides 7 and 9 (Fig. 3, D and F).
26283
D-Analogues of Antigenic Peptides '1
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BLEEDINGS
BLEEDINCS
FIG.3. Test in ELISA of the antibody response to the eight peptides 4-11 injected as SW-coupled peptides in BALB/c mice. Antisera were diluted 1:500 and allowed to react with the homologous peptides (A-H)or with H3 (Z-P).Anti-mouse I@ (H+L) conjugate (0) and anti-mouse IgG3 conjugate (). were diluted 1:5,000.Absorbance values were measured at 450 nm. The results represent the average absorbance values obtained in each group of mice ( n = 4). TABLE IV Recognition in competitive ELZSA of ZRGERA and ZRGERA analogues by mouse antibodies induced against homologous and analogue peptides Microtiter plates were coated with 2 p~ peptide conjugated to BSA by means of SPDP (carrier peptide molar ratio 1:lO) and allowed to react with mouse antisera raised against the homologous peptide (serum dilution 1:500) and preincubated with the various peptides used as inhibitors. Anti mouse IgG-peroxidase conjugate was diluted 1:5,000. Peptides used as inhibitor
1 4
Molar excess of inhibitor peptide required to inhibit 50% of the binding between anti-peptide antibodies and homologous antigens 1 4 5 6 7 8 9 1011
10 -
- 30 1010 1 0 1 0 10"" -
30 20 20
40
-
-
-
-
-
-
5 - - - - 6 7 - " " 8 10"- " " _ 9 20 - - - - - - - 10 6 - - - - - - - - 5 11 -, no detectable cross-reactivity (up to125 pg/ml peptide). "
"
By using in ELISA a conjugate specifically directed toward mouse IgG3, it was possible to detect a particular subpopulation of antibodies of the IgG3 isotype which was produced generally later during the course of immunization. This reactivity was not detected when anti-globulin conjugate was a mouse IgG (H+L) second antibody, presumably because the conjugate contained too few anti-IgG3 antibodies.
TABLE V Recognition in competitive ELZSA of H3 by mouse antibodies induced against peptide 4 and analoguepeptides Microtiter plates were coated with 2 pM peptide conjugated to BSA by means of SPDP (peptide-carrier ratio 1O:l) and allowed to react with mouse antisera raised against the homologous peptide (serum dilution 1:500) preincubated with various concentrations (0.25-125 pg/ml) of H3 used as inhibitor. The final reaction was revealed with rabbit anti-mouse IgG (H+L) conjugate (A) or with goat anti-mouse IgG3 (B) conjugated to horseradish peroxidase (working dilutions 1:5,000 and 1:7,000, respectively). Molar excesses were calculated as described under "Materials and Methods." Molar excess of H3 required to inhibit 50%of the Anti-mouse Ig reaction between antibodies against peptides 1 to 11 conjugate used to and the respective homologous peptides reveal reaction 1 4 5 6 7 8 9 1 0 1 1
A, Anti-total 0.1 Ig 0.05 0.4 0.4 -a - - - B, Anti-IgG3 - 1.0 4.4 -, no detectable cross-reactivity with H3 (up to 125 pg/ml). """
When the specificity of the IgG3 antibodies induced against the peptides 5-11 was tested indirect and competitive ELISA, it was found that these antibodies reacted in the same way with the various antigens as the other IgG subclasses except in the following two cases: 1)the IgG3 anti-peptide antibodies, especially those induced against peptides 7-11, reacted well and, in some cases, strongly with histone H3 (Fig. 3, GP).2) free peptides 4 and 11 but not the other peptide analogues inhibited the binding between H3 and IgG3 antibodies induced against peptide 11 (Fig. 44). In reciprocal tests, free peptides 4 and 11but not the other peptide analogues, inhib-
26284
0-Analogues of Antigenic Peptides
were the peptide analogues containing D-residues at positions 133,134, and 135recognized by antibodies against H3 or peptides 4, 5, and 6. The various partially D-analogues also differ significantly among themselves since they were not recognized by antibodies induced against the non-homologous PEPTIDES peptide analogues either in the direct or in the competition ELISA format where peptides are in the free form. Regarding the immunogenicity of the D-analogues, we found that each of the six D-analogues when coupled at the surface of SUV containing MPLA induced an immune response. The response, as revealed with anti-total IgG conju10 gate (which very likely detects IgG1, IgG2a, and IgG2b but not IgG3) was somewhat lower in the case of peptides 7 and 11 and,in general, of shorter duration in the case of all peptides containing D-residues. In contrast, IgG3 antibodies appeared later in the serum of immunized mice, reached a maximum in bleedings 4-5 and, in some cases (peptides 7, 9, and 111, remained at a significant level in bleeding 7, i.e. 70 days after the last injection. Thus the progression of IgG1, 2a, and 2b antibodies and that of IgG3 antibodies did not 01 follow the same pattern. Furthermore, antibodies of the IgG1, .I I 10 loo INX) 2a, and 2b subclasses were producedat a low level in response to injection of the D-enantiomer which initially led us to PEPTIDE (pdml) FIG. 4. Inhibition of the ELISA reaction between H3 (100 believe thatthis peptide was less immunogenic thanthe ng/ml) and IgG3 antibodies directed to peptide 11 ( A ) or to others. Overall, the results lead to some important conclusions. peptide 4 ( B )by increasing concentrations of peptides 4-11. Mouse antisera were diluted 1500 and anti-mouse IgG3 second an- First, it seems that the presence of IgG3 antibodies in the tibody 15,000. Absorbancevaluesweremeasured at 450 nm. The immunized mice can bemissedwhen an anti-mouse IgG molar excesses of peptides 4 and 11 overH3 required to inhibit 50% (H+L) peroxidase conjugate is used. It is important, therefore, of the antibody bindingwere about 300 in both cases. to determine which subclasses of IgG are actually detected by the indicator immunoconjugateand touse reagents that reveal ited the binding between H3 and IgG3 antibodies induced all antibodies with the same efficacy. against peptide 4 (Fig. 4 B ) . IgG3 antibodies raised against the Second, the high IgG3response in all mice immunizedwith all D-peptide 11reacted strongly with histone H3 in solution. the various peptides coupled to the surface of SUV is particAs shown in Table V, the parent protein H3 inhibited the ularly striking. Although we cannot directly compare the binding of anti-peptide 11 antibodies to peptide 11. Similar levels of reactivity of the antibodies of different subclasses results were not observed with IgG3antibodies raised against because the anti-mouse conjugates used in our ELISA proceother peptide analogues (Table VB) nor with IgG antibodies dure were different and that one arbitrary working dilution of other subclasses (Table VA). of 15,000 was chosen, the IgG3 response detected in immunized mice was significantly higher than usual. This finding DISCUSSION may result in part from the fact that we have used small Among the factors thought to affect the duration of the liposomes with surface-bound peptide and not a carrier proMPLA, and not immune response, persistence of the antigen in an appropriate tein for presenting peptides, andthat Freund's adjuvant, was used as adjuvant. Interestingly it has location is considered to play an important role (Gray and been reported that mouse IgG3 subclass antibodies predomiSkarvall, 1988).Thus one of the major problems in developing synthetic vaccines is to enhance the half-life of the peptides nate in humoral responses to protein antigens linked to the and increase the probability that they will interact with cells surface of liposomes whilethey constitute a small component of the immune system, in particular with dendritic cells. In of the humoral response to encapsulated protein (ThBrien et this study, we have used the liposome-peptide model devel- al., 1991) which is suggestive of a T-independent B-cell actioped previously (Friede et al., 1993) and explored the effect vation (Mc Kearn et al., 1982; Snapper and Mond, 1993). Third, when the specificity of antibody response is considof replacing L-amino acid residues by the corresponding Damino acids on both the duration and specificity of the im- ered, the antibody population of the IgG1, 2a, and 2b submune response. To thebest of our knowledge this is the first classes appears very specific in the sense that the antibodies detailed investigation of the influence of enantiomeric substi- of these subclasses induced against peptides 7, 8, 9, and 10, for instance, recognized only the homologous analogue peptutions on theimmunogenicity of peptides. As measured by the antigenic properties of the seven ana- tides and not the other partially modified D-analogues orthe logues produced in this study, the data suggest that neither cognate protein H3. In contrast,the IgG3 population contains the substitution of Arg13' and Arg'% by Lys residues nor the antibodies able to react both with the respective homologous replacement of Arg131 by a D-Arg changed the conformation peptides and with H3 on the plastic. Most interesting was the of the resulting peptides sufficiently to alter the binding of finding that the parent peptide as well as the protein H3, rabbit and mouse antibodies induced against H3 or against both in the free form, were also recognizedby anti-all Dpeptides 4, 5, and 6. In contrast, the substitution of G l P , peptide IgG3 antibodies. Reciprocally,IgG3 antibodies ink g " , and Ala"' by the respective enantiomers (peptides 7, duced against the parent peptide did recognize the homolo8, and 9) drastically altered the antigenicity of the modified gous peptide and H3 as well as the free all D-peptide. This peptides. In none of the presentations (eithercoupled to BSA cross-reactivity was only found between the parent peptide by means of SPDP or free in solution in inhibition assays) and the D-enantiomer (mirror image) but not when a single
0-Analogues of Antigenic Peptides D-amino acid was introduced in thesequence of the hexapeptide. The common structure between a peptide and itsmirror image is the peptide backbone (Guptasarma, 1992). Accordingly, the results suggest that the IgG3 antibodies recognize the backbone of the peptides 4 and 11 rather than the side chains. In this regard, it is noticeable that in this particular cross-reactivity with IgG3 antibodies to peptide 11,the parent peptide 4 andthe peptide 5 containing 2 lysine residues in replacement of arginine residues were not antigenically equivalent. It is well established that all L-amino acid polymers make right-handed a-helices whereas homopolymers of D-amino acid residues form left-handed ones. Therefore, the fact that IgG3 antibodies induced against the D-enantiomer reacted with the parent protein H3 and that IgG3 antibodies induced against the all L-peptide reacted with the free D-enantiOmer points to new possibilities of manipulating peptide antibody responses. Furthermore, the fact that it is only the IgG3 subclass that exhibits this cross-reactivity suggests that this class of antibody may bind to antigen in a unique manner possibly allowing a self-association which yields more effective binding, for example to thesurface of bacteria (Greenspan and Cooper, 1992). NMR studies of peptide-antibody complexes involving these analogues and monoclonal antibodies (Cung et al., 1991) may unravel the structural basis of this particular type of interaction. Acknowledgments-We are grateful to Drs. E. M. Moudrianakis (Baltimore) and E. Westhof (Strasbourg) for helpful advices, to Drs. P. Orlewski and M-T. Cung (Nancy) for attempts of molecular modeling, to Dr. M. John (Strasbourg) for help with CD measurements and to G . Sommermeyer for valuable technical assistance. REFERENCES Arenta. G., Burlin ame, R. F., Wan , B C., Love, W. E., and Moudrianakis, E. N. (1991)Proc. katl. A C ~ .sa. s.i. 88,10148-10152
8
26285
Amon, R., and Van Re enmortel, M. H. V. (1992)FASEB J. 6,3265-3274 Briand. J. P.. Muller. and Van Reeenmortel. M. H. V. (1985) . , J. Immunol. Methods 78,59-69 ' Briand, J. P., Barin, C., Van Regenmortel, M. H. V., and Muller, S. (1992)J. Immunol. Methods 168.255-265 Brown, F. (1990) Semin. Virol. 1.67-74 Butler, J. E., Li, L., Nessler, R., Joshi,K. S., Suter, M., Rosenberg, B., Chang, J.. Brown. W. R.. and Cantarero. L. A. (1992) . . J. Immunol. Methods 160. 71-90 Cung, M. T., Demange, P., Marraud, M., Tsikaris, V., Sakarellos, C., Papadouli, I., Kokla, A., and Tuutos, S. (1991)Biopolymers 31, 769-776 Fauchere, J. L., and Thurieau, C. (1992)Adv. Drug. Res. 23,127-159 Friede, M., Denery, S., Neimark, J., Kieffer, S., Gausepohl, H., and Briand, J. P. (1992)Peptide Res. 6,145-147 Friede, M., Muller, S., Briand, J. P., Van Regenmortel, M. H. V., and Schuber, F. (1993)Mol. Immunol. 30,539-547 Frisch, B., Muller, S., Briand, J. P., Van Regenmortel, M. H. V., and Schuber, F. (1991)Eur. J.Immunol. 21,185-193 Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986)Mol. Immunol. 24,709715 Geysen, H.M., Macfarlan, R., Rodda, S. J., Tribbick, G., Mason, T. J., and Shoofs, P. (1987)in Towards Better Carbohydrate Vaccines (Bell, R., and Torrigiani, G., eds) pp. 103-118,John Wiley and Sons,Chichester Gray, D., and Skarvall, H. (1988)Nature 336, 70-73 Greenspan, N. S.,and Cooper, L. J. N. (1992)Immunol. Today 13,164-168 Guptasarma, P. (1992)FEES Lett. 310,205-210 Hudson, D. (1988)J. Org. Chem. 63,617-624 Jung, G. (1992)Angew. Chem. Int. Ed. Engl. 31,1457-1459 King, D., Fields, C., and Fields, G. (1990)Int. J. Peptide Protein Res. 36, 255266 McKearn. J. P.. Paslav. J. W.. Slack., J... Baum., C... and Davie. J. M. (1982) . ~~, Immund. Rev. 64,5:23 . Milich, D. R. (1990)Semin. Immunol. 2,307-315 Milton, R. C., de L, Milton, S. C. F., and Kent, S . B. H. (1992)Science 266.
8..
I
'
1 Ads-1"" ddF( ""
Muller, S. (1988)in Synthetic Polypeptides as Antigem (Van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Plaue, S., eds) pp. 159-175, Elsevier, Amsterdam Muller, S., Himmelspach, K., and Van Regenmortel, M. H. V. (1982)EMBO J. 1,421-425 Muller, S., and Van Regenmortel, M. H. V. (1989)Methods Enzymol. 170, 251-263 Neimark, J., and Briand, J. P.(1993)Peptide Res. 6 , 219-228 Snapper, C. M., and Mond, J. J. (1993)Immunol. Today 14,15-17 Sorensen, K.,and Brcdbeck, U. (1986)J. Immunol. Methods 96,291-293 Therien, H. M., Shahum, E., and Fortin, A. (1991)Cell. Immuml. 138, 402413 Wade, D., Boman A., Wiihlin, B., Drain, C. M., Andreu, D., Boman, H. G., and Memifield, R. B. (1990)Proc. Natl. Acad. Sci. U.S. A . 87,4761-4765
