Differential Reactivity of Maleimide and Bromoacetyl Functions with

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-t...
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TECHNICAL NOTES Differential Reactivity of Maleimide and Bromoacetyl Functions with Thiols: Application to the Preparation of Liposomal Diepitope Constructs Philippe Schelte´, Christophe Boeckler, Benoıˆt Frisch,* and Francis Schuber Laboratoire de Chimie Bioorganique, UMR 7514 CNRS-Universite´ Louis Pasteur, Faculte´ de Pharmacie, 74 route du Rhin, 67400 Strasbourg-Illkirch, France . Received September 20, 1999; Revised Manuscript Received November 1, 1999

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as R- and -amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].

INTRODUCTION

Synthetic peptides representative of epitopes are useful for the development of new classes of subunit vaccines in infectious diseases or cancer (1-4). However, because of their weak immunogenicity, peptide antigens must generally be coupled to carriers such as proteins or synthetic polymers (5, 6) and administered in the presence of adjuvants. Alternatively, peptides can also be associated to particulate systems such as liposomes (7, 8) or immunostimulating complexes, e.g., ISCOMs (9). During these last years, our group has elaborated a strategy that elicits very potent and long-lasting immune responses against small peptide epitopes (10-13). It is based on liposomal constructs combining the peptide antigens and amphipathic adjuvants such as monophosphoryl lipid A (10-12) or the totally synthetic lipopeptide S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)cysteinyl-alanyl-glycine (Pam3CAG) (13). In our approach, the peptides are covalently coupled, via welldefined chemical steps (14, 15), to the surface of preformed small unilamellar vesicles containing the adjuvants. In general, the conjugation step was performed according to the method originally developed by Martin and Papahadjopoulos for the preparation of immunoliposomes (16). * To whom correspondence should be addressed. Phone: +33 388 67 68 72. Fax: +33 388 67 88 91. E-mail: [email protected].

Thus, the peptide extended at its N-terminus by a CG (i.e., cysteinyl-glycine in single letter code) spacer arm (i.e., HS-peptide) is reacted with a liposome containing a thiol-reactive phospholipid derivative such as phosphatidylethanolamine carrying a maleimide function (14, 16). This methodology presents the advantage to preserve the structural integrity of the peptide epitopes, and because of the lack of intrinsic immunogenicity of liposomes as carrier, it is appealing for the development of totally synthetic vaccination formulations. We have now designed a second generation of liposomebased carriers in which two different peptides, such as B and Th epitopes, are conjugated to vesicles with the aim to mimic and amplify a natural immune response. Diepitope constructs, which have been for example realized by co-linearization of B and Th epitopes in synthetic polypeptides, are considered to be the minimal built-in subunit vaccines (17, 18). Because of the vastly different role fulfilled by the B and Th epitopes in humoral immune responses, there is no a priori necessity for these epitopes to be covalently linked. We have therefore designed constructs with two structurally independent peptides conjugated to the surface of a same vesicle via different types of lipophilic anchors (19). The B epitope was classically conjugated to a phospholipid anchor (see above), whereas the Th epitope was coupled to a functionalized lipopeptide Pam3CAG (20). This latter anchor, which is a synthetic analogue of an Escherichia coli

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Figure 1. Liposome-based construct for immunization with synthetic peptides. The peptide carrying a cysteine residue, e.g., at its N-terminus, is conjugated to the surface of preformed SUV containing thiol-reactive lipophilic anchors. In the present example, the diepitope construct, HS-peptide (1) is conjugated to Pam3CAG functionalized with a maleimide group, and HS-peptide (2) to a phosphatidylethanolamine functionalized with a bromoacetyl group such as 2.

lipopeptide, was chosen for its immunoadjuvanticity and its known efficiency to elicit humoral as well as cellmediated immune responses against peptide antigens that are coupled to it (21). To that end, we have developed a chemical strategy that allows a controlled sequential coupling of two different HS-peptides to preformed liposomes containing thiol-reactive derivatives of phosphatidylethanolamine and Pam3CAG (19). On the basis of previous preliminary observations (14, 22) where maleimide and bromoacetyl functions were found to react differently with thiols, in aqueous media, we have used these groups to design our diepitope constructs (Figure 1). In the present publication, we describe our study on the comparative reactivity of maleimide and bromoacetyl groups with thiols in aqueous media as a function of pH. These studies were performed (i) with water-soluble functionalized model molecules, i.e., spacer arms that could also be coupled to lipophilic anchors incorporated into liposomes and (ii) with liposomes carrying at their surface these thiol-reactive functions. The aim was to define rate constants useful in predicting coupling rates and product distribution. We have also verified, under our most basic experimental conditions, the chemoselectivity of the bromoacetyl function by comparing its reactivity with thiols and other nucleophilic functions such as R- and -amino groups or imidazole that are present in peptides. Our results indicate that an important kinetic discrimination can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols, e.g., HS-peptides, are performed in aqueous media at pH 6.5 and 9.0, respectively. The present data also validate our strategy used to prepare liposomal diepitope constructs (Figure 1). EXPERIMENTAL SECTION

General. Reagent-grade solvents were used without further purification. Cholesterol (recrystallized in methanol), phosphatidylcholine (PC; egg yolk), phosphatidylglycerol (PG; transesterified from egg PC), and N-Racetyl-GK were from Sigma-Aldrich (L’Isle D’Abeau Chesnes, France). Fluorescamine (Fluram) and 5,5′dithiobis(2-nitrobenzoic acid) (DTNB) were from Fluka (L’Isle D’Abeau Chesnes, France). N-Benzoyl-GHL was from Calbiochem-Novabiochem AG (La¨ufelfingen, Switzerland). Silica gel 60 F 254 plates for thin-layer chromatography were purchased from Merck (Darmstadt, Germany). The peptide, CGIRGERA, was a kind gift from Dr. J.-P. Briand (CNRS-IBMC, Strasbourg, France). Compounds 3 and 4 were prepared according to our previously published procedures (14). 1H NMR were

recorded on Bruker AC-200 (200 MHz) and DPX 300 (300 MHz) spectrometers. Mass spectra were recorded on a VG ZAB-HF mass spectrometer. 2-Bromo-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)acetamide (1). This compound was synthesized in four steps starting from tri(ethylene glycol) with the same procedure as described previously by Frisch et al. (14): Rf 0.2 (CHCl3:CH3OH, 95:5). 1H NMR (200 MHz, CDCl3) δ 7.10 (s, 1H, NH), 3.87 (s, 2H, CH2Br), 3.76-3.44 (m, 12H, HOCH2, CH2OCH2, CH2N), 2.74 (s, 1H, OH). 13C NMR (50 MHz, CDCl3) δ 166.1, 72.4, 70.1 (2C), 69.3, 61.5, 39.7, 28.9. HRMS (FAB-) found for C8H16BrNO4, m/z 270.2 (M - H+); calcd for (M - H+) C8H16BrNO4, 270.12. Hexadecanoic Acid 3-((2-(2-(2-(2-(2-bromoacetylamino)ethoxy)ethoxy)acetylamino)ethoxy)hydroxyphosphoryloxy)-2-hexadecanoyloxypropylester (2). Compound 1 was oxidized into its corresponding carboxylic acid, which was then conjugated to DPPE according to procedures described in ref 14: Rf 0.34 (CHCl3: CH3OH:H2O, 60:15:1). 1H NMR (300 MHz, CDCl3: CD3OD, 1:1) δ 5.28-5.18 (m, 1H, CHCH2OP), 4.41-4.14 (m, 4H, CH2CHCH2OP, CHCH2OP), 4.02 (s, 2H, COCH2O) 3.99-3.43 (m, 12H, POCH2CH2N, POCH2CH2N, OCH2CH2), 3.87 (s, 2H, NCOCH2Br), 2.34-2.28 (m, 4H, CH2CO2), 1.68-1.52 (m, 4H, CH2CH2CO2), 1.26 (m, 48H, (CH2)24 ), 0.88 (t, J ) 6.0 Hz, 6H, CH3). 13C NMR (50 MHz, CDCl3) δ 173.7, 173.4, 171.3, 167.2, 70.8, 70.3, 69.9, 69.6, 64.5, 63.8, 62.8, 39.9, 34.3, 34.1, 31.9 (2C), 29.729.1 (23C), 25.0, 24.9, 22.7 (2C), 14.1 (2C). HRMS (FAB-) found for C45H87N2O12PBr, m/z 958.5 (M - H+); calcd for (M - H+) C45H87N2O12PBr, 959.07. Rate of Reaction of Thiol-Reactive Derivatives 1 and 3 with Thiols. An aqueous solution of 2-mercaptoethanol or L-cysteine or peptide CGIRGERA (40 µL, 200 nmol) was added to 450 µL of 50 mM sodium phosphate buffers (pH 6.0, 6.5, 7.0 or 7.5) or 50 mM sodium borate buffers (pH 8.0, 8.5 or 9.0) maintained at 25 °C (thermostat). At time zero, an aqueous solution of 1 or 3 (10 µL, 100 nmol) was added and the reaction progress was followed by determining at given times the residual thiols (23) on 40 µL aliquots with 1 mL of Ellman’s reagent (DTNB 2 mM and EDTA 1 mM in 0.2 M sodium phosphate buffer, pH 7.27). The absorbance was read at 412.5 nm ( ) 14 150 M-1 cm-1). All the given data have been corrected for the spontaneous dimerization of the thiols, which was estimated in parallel under the same experimental conditions, but in the absence of 1 and 3. Reaction of Thiols with Thiol-Reactive Functionalized Phosphatidylethanolamines 2 and 4 Incorporated into Liposomes. Small unilamelar vesicle (SUV) were prepared from PC, PG, 2, or 4 and cholesterol (molar ratio 65:25:10:50) as previously described (10). Briefly, the lipids in chloroform were dried under high

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vacuum and resuspended to 5 µmol of lipid/mL in 10 mM Hepes buffer, pH 6.5, containing 145 mM NaCl or 50 mM sodium borate, pH 9.0, containing 105 mM NaCl, vortexed during 5 min and sonicated under argon at room temperature in a sonicator bath for 20 min. Then the suspension was extruded six times on 0.1 µm Nucleopore membranes. The mean diameter of the vesicles was determined by a photon correlation spectroscopy technique, using a Malvern Autosizer. The liposome concentration was determined by assaying the inorganic phosphate (24). At time zero, to a liposome suspension (700 µL, 140 nmol of surface-accessible thiol-reactive functions), maintained at 25 °C, was added an aqueous solution of the peptide CGIRGERA (30 µL, 280 nmol). The reaction progress was measured at given times by determining the residual thiols on 50 µL aliquots as described above. The data have been corrected for the spontaneous dimerization of the thiols which was estimated in parallel under the same experimental conditions, but in the absence of 2 and 4. Rate of Reaction of 1 with Peptidic Amine Functions. An aqueous solution of N-R-acetyl-GK (40 µL, 200 nmol) was added to 450 µL 50 mM sodium borate buffer (pH 9.0). An aqueous solution of 1 (10 µL, 100 nmol) was added at time zero. The reaction was thermostated at 25 °C, and its progress was measured at given times by measuring the residual primary amines with the fluorometric assay using fluorescamine (25). Briefly, 5-40 µL of the test solution containing from 0 to 100 nmol of amines was added to 1.5 mL of 50 mM sodium borate buffer, pH 9.0, followed, under rapid stirring, by 500 µL of fluorescamine in dioxane (30 mg/100 mL). The fluorescence was measured at 475 nm (excitation 390 nm). The primary amines were quantified using a standard curve obtained with of N-R-acetyl-GK. Reaction of the Bromoacetyl Function with a Peptidic Imidazole Residue at pH 9.0. To determine the reactivity of an imidazole moiety present in a peptide, SUVs were prepared in 50 mM sodium borate buffer, pH 9.0, from 5 µmol of lipid/mL containing PC, PG, 2, and cholesterol in the molar ratio of 65:25:10:50. To a suspension of vesicles (800 µL, 200 nmol of bromoacetyl function), an aqueous solution of peptide N-benzoyl-GHL (80 µL, 400 nmol) was added. After 90 min at 25 °C, the liposomes were dialyzed 2 × 12 h against 1 L of 50 mM sodium borate buffer, pH 9.0. One hundred microliter aliquots were then added to 100 µL of 12 N HCl and hydrolyzed 12 h at 110 °C followed by neutralization with 6 N NaOH. The conjugation of the peptide to the liposomes was assessed by determining the amines with the fluorescamine fluorometric assay (see above). Liposomes without 2 were used as control and known quantities of N-benzoyl-GHL as standard. Competitive Reaction between Thiol and Amine Functions of CGIRGERA with the Bromoacetyl Function of 1. A solution (40 µL) containing 100 nmol of the peptide CGIRGERA was added to 450 µL 50 mM sodium borate buffer, pH 9.0. At time zero, an aqueous solution of 1 (10 µL, 50 nmol) was added and the progress of the reaction, at 25 °C, was measured by assaying at given times on 40 µL aliquots (i) the residual thiols as described above (Ellman’s reagent) and (ii) the residual primary amines with the fluorescamine assay (see above, using CGIRGERA as standard). The given data take into account the dimerization of the thiol that was measured in parallel under the same experimental conditions but in the absence of 1. Rate Constants Determination. The apparent rate constants between the thiols and the thiol-reactive func-

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Figure 2. Structure of the thiol-reactive derivatives 1 and 3 and their DPPE derivatives 2 and 4.

tions 1-4 were determined using a standard secondorder reaction rate expression and linear-regression programs (GraphPad Prism and Microsoft Excel). RESULTS AND DISCUSSION

To investigate the comparative reactivity of maleimide and bromoacetyl functions with thiols under conditions that could be exploited for the coupling, in aqueous media, of HS-peptides to preformed liposomes carrying such reactive groups at their surface, we have first prepared the water-soluble model heterobifunctional molecules 1 and 3 (Figure 2). These polyoxyethylenebased flexible molecules could also be used to produce 2 and 4, i.e., thiol-reactive dipalmitoylphosphatidylethanolamine (DPPE) derivatives that were incorporated into the (phospho)lipid composition of liposomes. The chemistry used to prepare molecules 1-4 has been adapted from our previous work in this domain (14). Reactivity of the Thiol-Reactive Moieties in 1 and 3 with Different Thiols as a Function of pH. The aim of our study was to couple two different HS-peptides, each on a specific anchor, to the surface of the same preformed liposome (Figure 1). It was therefore of importance to define experimental conditions (pH, temperature, and time) allowing a sequential conjugation of the peptides on two different thiol-reactive lipophilic anchors, which would be compatible with the integrity of the vesicles used in the immunization experiments. We have therefore first studied the rate of reaction, in aqueous media, of the bromoacetyl derivative 1 and the maleimide derivative 3 with 2-mercaptoethanol, L-cysteine, and CGIRGERA, an antigenic model peptide (10, 11) which carries a cysteine residue at its N-terminus. The reactions were performed at 25 °C and at fixed pH values, at a molar ratio of 2 in favor of the thiol. Reaction progress was determined by measuring residual-free thiols with Ellman’s reagent. Controls, under these different reaction conditions, allowed the determination of the spontaneous oxidation of thiols (e.g., dimerization) and accordingly the correction of the experimental data. At pH 9.0 and 25 °C, the spontaneous oxidation [kdim (M-1 s-1)] was 0.054 ( 0.003 for mercaptoethanol, 0.113 ( 0.01 for cysteine,

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Figure 3. Reaction between the thiol-reactive derivatives 1-4 with 2-mercapthoethanol, cysteine, and the peptide CGIRGERA at different pH values. The reactions were performed at 25 °C in the presence of a 2-fold molar excess of thiol (see text) and reaction progress was determined by measuring residual thiols with Ellman’s reagent. Reaction of 2-mercapthoethanol with 3 (panel A) and 1 (panel B), of cysteine with 3 (panel C) and 1 (panel D) and of CGIRGERA with 1 and 3 (panel E) and with liposomal 2 and 4 (panel F). (Panels E and F) The numbers in parentheses designate the thiol-reactive reagents 1-4 used in these experiments.

and 0.466 ( 0.03 for CGIRGERA. At pH 6.5, kdim was 800 and 0.38 ( 0.02 M-1 s-1, respectively). A similar difference was observed in the case of cysteine (k ) >330 and 0.21 ( 0.04 M-1 s-1, respectively) and 2-mercaptoethanol (k ) 101 ( 0.3 and 0.052 ( 0.02 M-1 s-1, respectively). These differences of reactivity between the thiol-reactive functions are large enough to perform a preferential coupling, at pH 6.5, of an HS-peptide to a maleimide derivative even in the presence of a bromoacetyl function which reacts much faster at basic pH. Thus, a sequential coupling of two HS-peptides can be envisaged, first on a maleimide derivative at pH 6.5 followed by a coupling to a bromoacetyl derivative at, e.g., pH 9.0 under experimental conditions that respect the integrity of liposomes. Reaction of the Peptide CGIRGERA with the Thiol-Reactive Functionalized Phosphatidylethanolamines 2 and 4 Incorporated into Liposomes. In solution, we have found an important difference of reactivity, at pH 6.5, between the bromoacetyl 1 and maleimide 3 with the terminal Cys residue of CGIRGERA (Figure 3E). Next, we wanted to assess the extent of the differential reactivity of these functions when present at the surface of liposomes. The functionalized spacer-arms were thus conjugated to DPPE to give, respectively, the thiol-reactive lipophilic anchors 2 and 4 (Figure 2), which could be incorporated into liposomes. SUVs composed of PC, PG, 2 or 4, and cholesterol (molar ratio 65:25:10:50) were prepared (average diameter 100 nm) and incubated in the presence of 2 equiv of CGIRGERA/reactive group. At pH 6.5, the reaction with 4 was very fast, whereas with 2, the rate of coupling was much lower (Figure 3F) and the difference in rate constants between the two conjugation reactions was estimated to

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be about 2 orders of magnitude (k ) >54 ( 6 and 0.28 ( 0.01 M-1 s-1, respectively). As expected, the peptide reacted with the bromoacetyl derivative 2 at higher pH (Figure 3F). The difference of reactivity between the maleimide and bromoacetyl functions with the HS-peptide is affected by their presence on liposomes; thus, we find 2-orders of magnitude difference instead of 3 in solution. Interestingly, only the reactivity of the maleimide moiety is affected when present at the surface of the vesicles, whereas the bromoacetyl function reacts at similar rates in solution or exposed on liposomes. It seems that the reaction of the maleimide with the peptide is so fast that when present at the surface of liposomes the lower diffusion rate of the vesicles could come into play. This difference of reactivity, at pH 6.5, between the maleimide and bromoacetyl functions, although diminished when present at the surface of liposomes, is still important enough to permit the preparation of the diepitope constructs (Figure 1). In principle, this difference could be optimized further when needed by reacting the maleimide at lower pH values and/or by using in the first step equimolar amounts of HS-peptide and liposomal maleimide derivative such as 4. We have used this latter strategy which is kinetically more controllable and thus avoids “overshooting” by a slow reaction of the excess peptide with the bromoacetyl derivative (e.g., 2) (19). Reactivity of the Bromoacetyl Derivative 1 with Other Functions at Basic pH. To be able to use a bromoacetyl functionalized lipophilic anchor to conjugate a HS-peptide to the surface of preformed liposomes, we wanted to make sure that the coupling reaction was still chemoselective, at the basic pH used, for the thiol group present at its N-terminus. We thus controlled the reactivity of 1, at pH 9.0, with other nucleophilic moieties likely to be present in peptides such as the N-terminal function, -NH2 of lysine or imidazole of histidine residue. The reactivity of the -NH2 function of lysine was tested with the dipeptide of N-R-acetyl-GK. When 1 was reacted with 2 equiv of this peptide, no disappearance of amine functions was measurable over a period of 60 min, at 25 °C, as assessed by the fluorescamine assay. Under the same conditions, to test the reactivity of a N-terminal function of a peptide, we used 2 equiv of CGIRGERA and measured in parallel the reactivity of the thiol group and of the amine residues, respectively, with the Ellman’s reagent and fluorescamine. The results indicated over the same time a complete loss of reactive thiols and no reaction of the N-terminus. To test the reactivity of an imidazole moiety of a peptide with the bromoacetyl group at pH 9, we used the tripeptide N-benzoyl-GHL, which contains an imidazole side chain whose N-terminus is protected. This peptide (2 equiv) was added to liposomes containing 2 and after 90 min at 25 °C an extensive dialysis was performed. After complete hydrolysis of the liposomal preparation, analysis revealed no coupling of the peptide to the vesicles. Altogether, these results indicate that the bromoacetyl function of 1 and 2 presents a high chemoselectivity for peptidyl thiol groups; i.e., under experimental conditions where the reaction of thiols is essentially complete, no reaction with peptidic R- or -NH2 and imidazole functions could be detected. This is in excellent agreement with previous reports showing that of the nucleophilic groups present in proteins/peptides thiols are modified most readily by electrophilic reagents, whereas amino groups are less reactive by 2-3 orders of magnitude and imidazole groups of histidine are even less reactive (27 and references therein). Therefore, it is most likely that,

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in our constructs, a HS-peptide will be conjugated via the unique formation of a thioether bond between its N-terminal thiol and the bromoacetyl group. CONCLUSION

We have demonstrated the existence of a great difference of reactivity, in aqueous media, of thiols carried by peptides with maleimide and bromacetyl functions. The reaction with maleimide is much faster at pH 6.5 than with the bromoacetyl group; the conjugation to the latter becomes exploitable only at higher pH values (e.g., pH 9.0) while remaining chemoselective for the thiol functions. We have applied this differential reactivity to conjugate sequentially, to the surface of a same preformed vesicle, two different HS-peptide epitopes on predefined and different thiol-reactive lipophilic anchors. Thus, using a B epitope originating from a Streptococcus mutans cell surface adhesin and a “universal” Th epitope from tetanus toxin, we have prepared a liposomal diepitope construct (19) and showed that this synthetic formulation when administered i.p. to BALB/c mice induced a highly intense, anamnestic, and long-lasting (over 2 years) immune response. This differential reactivity of maleimide and bromoacetyl functions could be exploitable with any other construct where sequential coupling of different thiol carrying ligands is of importance. LITERATURE CITED (1) Arnon, R., and Horwitz, R. J. (1992) Synthetic peptides as vaccines. Curr. Opin. Immunol. 4, 449-453. (2) Arnon, R., and Van Regenmortel, M. H. V. (1992) Structural basis of antigenic specificity and design of new vaccines. FASEB J. 6, 3265-3274. (3) Brown, F. (1994) Synthetic peptides and purified antigens as vaccines. Int. J. Technol. Assess. Health Care 10, 161166. (4) Ben Yedidia, T., and Arnon, R. (1997) Design of peptide and polypeptide vaccines. Curr. Opin. Biotechnol. 8, 442-448. (5) Jackson, D. C., O’Brien-Simpson, N., Ede, N. J., and Brown, L. E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15, 1697-1705. (6) Tam, J. P. (1996) Recent advances in multiple antigen peptides. J. Immunol. Methods 196, 17-32. (7) Alving, C. R., Koulchin, V., Glenn, G. M., and Rao, M. (1995) Liposomes as carriers of peptide antigens: induction of antibodies and cytotoxic T lymphocytes to conjugated and unconjugated peptides. Immunol. Rev. 145, 5-31. (8) Gregoriadis, G., Gursel, I., Gursel, M., and McCormack, B. (1996) Liposomes as immunological adjuvants and vaccine carriers. J. Controlled Release 41, 49-56. (9) Barr, I. G., Sjolander, A., and Cox, J. C. (1998) ISCOMs and other saponin based adjuvants. Adv. Drug Delivery Rev. 32, 247-271. (10) Frisch, B., Muller, S., Briand, J. P., Van Regenmortel, M. H. V., and Schuber, F. (1991) Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen. Eur. J. Immunol. 21, 185-93. (11) Friede, M., Muller, S., Briand, J. P., Van Regenmortel, M. H. V., and Schuber, F. (1993) Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid A. Mol. Immunol. 30, 539-547. (12) Friede, M., Muller, S., Briand, J. P., Plaue, S., Fernandes, I., Frisch, B., Schuber, F., and Van Regenmortel, M. H. V. (1994) Selective induction of protection against influenza virus infection in mice by a lipid-peptide conjugate delivered in liposomes. Vaccine 12, 791-797. (13) Fernandes, I., Frisch, B., Muller, S., and Schuber, F. (1997) Synthetic lipopeptides incorporated in liposomes: In vitro stimulation of the proliferation of murine splenocytes and in

Technical Notes vivo induction of an immune response against a peptide antigen. Mol. Immunol. 34, 569-576. (14) Frisch, B., Boeckler, C., and Schuber, F. (1996) Synthesis of short polyoxyethylene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes. Bioconjugate Chem. 7, 180-186. (15) Boeckler, C., Frisch, B., Muller, S., and Schuber, F. (1996) Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes. J. Immunol. Methods 191, 1-10. (16) Martin, F. J., and Papahadjopoulos, D. (1982) Irreversible Coupling of Immunoglobulin Fragments to Preformed Vesicles. J. Biol. Chem. 257, 286-288. (17) Goodman-Snitkoff, G., Eisele, L. E., Heimer, E. P., Felix, A. M., Anderson, T. T., Fuerst, T. R., and Mannino, R. J. (1990) Defining minimal requirements for antibody production to peptide antigen. Vaccine 8, 257-262. (18) Brumeanu, T. D., Casares, S., Bot, A., Bot, S., and Bona, C. A. (1997) Immunogenicity of a contiguous T-B synthetic epitope of the A/PR/8/34 influenza virus. J. Virol. 71, 54735480. (19) Boeckler, C., Dautel, D., Schelte´, P., Frisch, B., Wachsmann, D., Klein, J. P., and Schuber, F. (1999) Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes. Eur. J. Immunol. 29, 2297-2308. (20) Boeckler, C., Frisch, B., and Schuber, F. (1998) Design and synthesis of thiol-reactive lipopeptides. Bioorg. Med. Chem. Lett. 8, 2055-2058. (21) Bessler, W. G., Baier, W., van den Esche, U., Hoffmann, P., Heinevetter, L., Wiesmu¨ller, K.-H., and Jung, G. (1997)

Bioconjugate Chem., Vol. 11, No. 1, 2000 123 Bacterial lipopeptides constitute efficient novel immunogens and adjuvants in parenteral and oral immunization. Behring Inst. Mitt. 98, 390-399. (22) Kichler, A., Remy, J. S., Boussif, O., Frisch, B., Boeckler, C., Behr, J. P., and Schuber, F. (1995) Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids. Biochem. Biophys. Res. Commun. 209, 444450. (23) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5, 5′-dithiobis(2-nitrobenzoı¨c acid): a reexamination. Anal. Biochem. 94, 75-81. (24) Rouser, G., Fleischer, J., and Yamamoto, A. (1970) Twodimensional thin layer chromatographic separation of plant lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494-496. (25) Bo¨hlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 155, 213-220. (26) Nieslanik, B. S., and Atkins, W. M. (1998) Contribution of linear free energy relationships to isozyme- and pH-dependent substrate selectivity of glutathione S-transferases: comparison of model studies and enzymatic reactions. J. Am. Chem. Soc. 120, 6651-6660. (27) Dienys, G., Sereikaite´, J., Gave`nas, G., Kvederas, R., and Bumelis, V. A. (1998) Cross-linking of protein subunits by 1,3,5-triacryloyl-hexahydro-s-triazine. Bioconjugate Chem. 9, 744-748.

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