Construction of Immunogenic Conjugates - American Chemical Society

T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. ... Formulation of an effective immunogen that c...
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Bioconjugate Chem. 2005, 16, 812−819

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A Peptide Carrier with a Built-In Vaccine Adjuvant: Construction of Immunogenic Conjugates Dimitrios Krikorian, Eugenia Panou-Pomonis, Chryssa Voitharou, Constantinos Sakarellos, and Maria Sakarellos-Daitsiotis* Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, 45110 Ioannina, Greece. Received December 13, 2004; Revised Manuscript Received April 18, 2005

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1β(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1β without possessing any of the pro-inflammatory properties of IL-1β was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOCn, formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593599), was also positioned in the carboxy terminus of SOCn as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sjo¨gren’s Syndrome, respectively, were coupled to the Lys-NH2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/ incomplete Freund’s adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund’s adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.

INTRODUCTION

Formulation of an effective immunogen that could elicit a potent immune response should incorporate B cell epitopes that mimic the three-dimensional conformation of the antigen and T cell epitopes resulting from the processed protein, both on the same carrier that links the major histocompatibility complex with the T cell receptor (1, 2). To achieve enhanced and prolonged humoral and cellular immune response, potent immunogens must be also administered in combination with adjuvants, e.g. incomplete Freund’s, CRL1005, QS21, Montanide ISA-51, etc., although the precise mechanism of their action remains unclear, and severe skin toxicities, in some cases, have been reported. The only adjuvants currently approved by the Food and Drug Administration for use with human vaccines are alluminum salts, which, however, have low potency and problems associated with their formulation. To address this problem, adjuvants with low toxicity profiles as for example the granulocyte monocyte colony stimulating factor have been tested (3, 4). It has been also proposed that T cells provide “help” to B cells under genetic control. Recently, a number of so-called “promiscuous” T cell epitopes from tetanus toxin (TT), measles virus, or E6 transforming protein have been reported to be universally immunogenic (5, 6). On the basis of the reported issues, an ideal immunogenic conjugate, and consequently an effective human vaccine, should combine specific B and T cell epitopes, adjuvants, and “promiscuous” T cell epitopes. * Corresponding author. Fax: +30-26510-98770, Tel: +3026510-98386, e-mail: [email protected].

The rational design of such a multivalent conjugate as vaccine candidate requires a synthetic carrier or template on which different peptides with various functionalities could be anchored. Development of synthetic carriers for anchoring immunogenic peptides and producing specific immune responses has been the stimulus of intensive efforts. Among others the lysine core matrix, the Pam3Cys-Ser (a palmitoyloxy lipopeptide), and the templateassembled synthetic protein (TASP)1 deserve to be mentioned (7-9). A new class of Sequential Oligopeptide Carriers, (SOCn), for anchoring antigenic/immunogenic peptides has been successfully applied in our laboratory. The carrier, formed by the repetitive Lys-Aib-Gly moiety, was designed to display a predetermined 3D structure, so that the attached peptides could obtain a defined spatial 1 Abbreviation: Ac O, acetic anhydride; Alloc, allyloxycarbo2 nyl; Boc, tert-butyloxycarbonyl; Bzl, benzyl; CD, circular dichroism; CFA/IFA, complete/incomplete Freund’s adjuvant; CMC, critical micelle concentration; DCM, dichloromethane; DIEA, disopropylethylamine; DMF, dimethylformamide; ESI-MS, electron spray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; HF, hydrogen fluoride; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; IL1β, interleukin-1β; LC, liquid chromatography; MBHA, 4-methylbenhydrylamine resin; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SLE, systemic lupus erythematosus; SOCn, Sequential Oligopeptide Carrier; SPPS, stepwise solidphase procedure; SS, Sjogren’s Syndrome; TASP, templateassembled synthetic protein; TBTU, 2-(1H-benzotriazol-1-y1)1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TMB, 3,3′,5,5′-tetramethylbenzidine; Tos, tosyl; TT, tetanus toxin.

10.1021/bc049703m CCC: $30.25 © 2005 American Chemical Society Published on Web 06/10/2005

Carrier Immunostimulatory Effect

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Figure 2. Schematic representation of conjugates Ac-IL-1βSOC4-Sm4-TT-NH2 (2) and Ac-IL-1β-SOC4-[Ac2-(La/SSB)2]-TTNH2 (3), where Sm: PPGMRPP, La/SSB: Ac-ANNGNLQLRNKEVTWEVLEG (289-308), IL-1β: Ac-VQGEESNDK (163171), and TT: YSYFPSV (593-599).

Figure 1. Schematic representation of a multivalent peptide carrier that incorporates an adjuvant, IL-1β(163-171), a promiscous T cell epitope, TT(593-599), and specific B and T cell epitopes anchored to the Lys-NN2 groups of the sequential oligopeptide carrier (Lys-Aib-Gly)4, SOC4.

orientation. Conformational analysis showed that SOCn adopt a distorted 310 helicoid structure, while the coupled epitopes preserve their original “active” structure. It has been concluded that the helicoid structure of SOCn helps the reconstitution and/or mimicking of the native forms of the epitopes, so that potent immunogens could be generated. SOCn-conjugates induced high titers of specific antibodies recognizing the priming peptides, as well as the intact proteins, but not the carrier alone (10-14). With the aim of contributing to the development of a carrier that could help to augment and improve the character and the quality of the humoral and cellular immune response, the SOCn carrier was elongated from the amino-terminus by the IL-1β fragment VQGEESNDK (163-171) as adjuvant. It has been reported that IL-1β(163-171) is devoid of all pro-inflammatory effects of IL1β but maintains the immunostimulatory activity of the intact cytokine (15, 16). A “promiscuous” T cell epitope deriving from TT, YSYFPSV (593-599), was also covalently attached to the carboxy-terminal group of SOCn, as universal immunogen (17). B and T cell epitopes derived (i) from the Sm and (ii) the La/SSB antigens were coupled to the Lys-NH2 groups of the carrier and the obtained conjugates were administered in animals without adding any adjuvant to test the induction of specific antibodies. The schematic representation of a multivalent peptide vaccine that incorporates an adjuvant IL-1β(163171), a promiscuous T-cell epitope TT(593-599), and specific B and T cell epitopes is illustrated in Figure 1. Epitope mapping of the Sm autoantigen revealed that the sequence PPGMRPP, found in three copies in the protein, represents the major B cell epitope of Sm, against which is directed the majority of autoantibodies in SLE patients (18). In this regard anti-Sm antibodies are considered as a disease marker for SLE (19, 20). Four copies of the PPGMRPP dominant B cell epitope of Sm were covalently anchored to the carrier and the potential of the reconstituted Sm conjugate, Ac-IL-1β-SOC4-Sm4TT-NH2 (2) (Figure 2) to elicit specific immune response without usage of any adjuvant was examined. La/SSB autoantigen is one of the molecular targets of humoral autoimmunity primarily in SS patients (21). In previous studies we have mapped the location of four B cell antigenic determinants of La/SSB (22), and we have investigated their ability to induce T cell responses against the La/SSB protein after animal injection (23). It was found that the sequence ANNGNLQLRNKEVTW-

EVLEG corresponding to the region 289-308 of La/SSB is an immunodominant T-cell and a minor B cell epitope. Two copies of the La/SSB (289-308) epitope were covalently linked to the 1st and 4th Lys-NH2 group of SOC4 to create a mimic of La/SSB. Animal immunization experiments were performed to validate the efficacy of the Ac-IL-1β-SOC4-[Ac2, (La/SSB)2]-TT-NH2 (3) conjugate (Figure 2) to induce high titers of specific antibodies without utilizing any adjuvant. The produced potent humoral immune response of both conjugates was compared with that obtained when each one of them was injected following the complete/incomplete Freund’s adjuvant protocol. Our data suggest that the presented carrier could be employed in engineering potent immunogenic conjugates as human vaccine candidates. EXPERIMENTAL PROCEDURES

Synthesis of the Ac-IL-1β-SOC4-Ac4-TT-NH2 Carrier (1). The synthesis of 1, where SOC4 is (K-Aib-G)4, was carried out manually by the SPPS procedure (2426) using the MBHA resin (3 g, 0.13 mmol/g capacity). Lysine was introduced as Boc-Lys(Fmoc)-OH, serine as Boc-Ser(Bzl)-OH, tyrosine as Boc-Tyr(2,6-di-Cl-Bzl)-OH, and aspartic acid and glutamic acid as Boc-Asp(Obzl)OH and Boc-Glu(Obzl)-OH, respectively. Synthetic procedure starts with the step by step couplings, to the resin, of the protected residues (Boc/Bzl) corresponding to the TT(593-599) fragment. The protocol of the synthesis was the following: (i) deprotection with 40% TFA/DCM (1 × 2 min, 1 × 13 min); (ii) washings DCM (3 × 1 min), MeOH (3 × 1 min), DCM (3 × 1 min); (iii) neutralization with 7% DIEA (2 × 2 min); (iv) washings DCM (3 × 1 min), MeOH (3 × 1 min), DCM (3 × 1 min); (v) couplings with three equivalent amino acid derivatives, 2.9 equiv of TBTU, 3 equiv of HOBt, 9 equiv of DIEA in DCMDMF 4:1 or 1:4 (V/V) mixture depending on the solubility of Boc-amino acid derivatives (2 h); (vi) washings DCM (3 × 1 min), MeOH (3 × 1 min), DCM (3 × 1 min); (vii) ninhydrin assay (27). A similar protocol was applied for the sequential propagation of the Boc-K (Fmoc)-Aib-Gly moiety until the formation of the tetrameric SOC4, oligopeptide. The IL-1β(163-171) fragment was inserted step by step following the Boc/Bzl strategy to obtain the protected carrier on the resin. The NR-terminal Boc group (Boc-V) of the carrier was cleaved by 40% TFA in DCM and Val was NR-acetylated with Ac2O (30 equiv of Ac2O/pyridine, 20 min). Fmocprotective groups of Lys-NH2 were removed by 20% piperidine in DMF (1 × 2 min, 1 × 15 min, washings DCM, MeOH, DCM) followed by N-acetylation with Ac2O. The peptide was cleaved from the resin support by liquid HF in the presence of phenol and anisole (HFphenol-anisole ) 10 mL:1 g:1 mL) for 30 min at -8 °C and 1.5 h at 0 °C and extracted with 2 M aqueous acetic

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acid. The crude peptide (yield 90%) was purified by RPHPLC using a Supelco Discovery C18 semipreparative column (250 mm × 10 mm, 5 µm, 300 Å). The purified peptide (yield 57%) was analyzed by RP-HPLC (tR ) 15.41 min), LC and ESI-MS (M + H+ calculated/found 3181.5/3181.6). Synthesis of the Ac-IL-1β-SOC4-Sm4-TT-NH2 Conjugate (2). Initially was synthesized, by the SPPS procedure, on the resin as the protected fragment of the carrier comprising TT and SOC4, as already described for the synthesis of 1, except that the fourth lysine was introduced as Alloc-K(Fmoc). After removal of the Fmoc protective groups from the Lys-NH2 groups by 20% piperidine in DMF, synthesis of the Sm epitopes was carried out (Boc/Bzl) by the simultaneous attachment of each residue in four copies and afterward the NR-Alloc group of Lys was eliminated under hydrostannolytic cleavage [PbCl2(PPh3)2/Bu3SnH/AcOH in DCM (0.03 equiv of PbCl2(PPh3)2, 3 equiv of Bu3SnH, and 3 equiv of AcOH for 30 min, washings DCM, MeOH, DCM] (28, 29), and the N-terminal sequence of IL-1β(163-171) was coupled step by step to the carrier. Arginine was introduced as Boc-Arg(Tos)-OH, while Met was used without side protection as Boc-Met-OH. Dimethyl sulfide was added to each cycle of Boc-removal (TFA/DCM/DMS: 40/59/1) after coupling of Boc-Met-OH to avoid oxidation of Met side chain. The peptide was cleaved from the resin by liquid HF in the presence of p-cresol and thiocresol as described for 1. The crude material (yield 86%) was dialyzed against water (yield 33%) and purified by RPHPLC using a Supelco Discovery C18 semipreparative column as for peptide 1. The purified peptide (yield 24%) was analyzed by RP-HPLC (tR ) 10.12 min), LC and ESIMS (M + H+ calculated/found 5940.95/5940.90). Synthesis of the Ac-IL-1β-SOC4-[Ac2, (La/SSB)2]TT-NH2 Conjugate (3). Synthesis of the protected fragment of the carrier, incorporating TT(593-599) and SOC4, was performed as for 1, except that lysines at the 1st and 4th position of SOC4 were introduced as AllocK(Fmoc) and Boc-K(Fmoc), respectively, while lysines at the 2th and 3rd position were inserted as Boc-K(Ac)-OH. After cleavage of the Fmoc protective group of lysine side chains, the synthesis of the La/SSB (289-308) epitope was carried out by the simultaneous coupling of each residue in two copies following the Boc methodology, except that lysine within the epitope sequence was introduced as Alloc-Lys(Fmoc)-OH. The NRH2 terminus of the epitopes was acetylated, while after removal of the NR-Alloc protective group of the carrier it was elongated to construct the IL-1β(161-173) N-terminal segment. The peptide was cleaved from the resin by liquid HF in the presence of phenol and anisole as described for 1. The crude material (yield 91%) was dialyzed against water (yield 70%) and purified by RP-HPLC using a Supelco Discovery C18 semipreparative column as for peptide 1. The purified peptide (yield 18%) was analyzed by RPHPLC (tR ) 17.10 min), LC and ESI-MS (M + H+ calculated/found 7619.43/7619.65). Dialysis. Dialysis tubes, whice are cellulose membranes benzoylated in order to reduce the pore size, with a molecular mass cutoff of ∼1500, were obtained from Sigma (Deisenhofen, Germany). They were supplied in water pack containing 0.002% 1-hydroxypyridine-2thione as preservative, and they were activated by heating in a solution of 0.1 M NaHCO3 and 0.001 M ethylenediaminetetraacetic acid to just under boiling point. LC-ESI-MS. LC-ESI-MS of Ac-IL-1β-SOC4-Ac4-TTNH2 (1), Ac-IL-1β-SOC4-Sm4-TT-NH2 (2), and Ac-IL-1β-

Krikorian et al.

SOC4-[Ac2-(La/SSB)2]-TT-NH2 (3) was performed using a Supelco Discovery C18 (250 mm × 4.6 mm, 5 µm, 300 Å) reverse phase column with a Waters instrument equipped with a Waters 616 pump and a Waters 2487 Dual λ absorbance detector. Eluent A was 0.1% TFA in water, and eluent B was 0.1% TFA in acetonitrile. A linear gradient for Ac-IL-1β-SOC4-Ac4-TT-NH2 (1) and AcIL-1β-SOC4-Sm4-TT-NH2 (2) was 20% to 50% acetonitrile in 0.1% TFA at a flow rate of 1 mL/min and for Ac-IL1β-SOC4[Ac2-(La/SSB)2]-TT-NH2 (3) was 20% to 40% acetonitrile in 0.1% TFA at a flow rate of 1 mL/min. RP-HPLC. RP-HPLC purification was carried out on a Waters 4000 semipreparative instrument using a Supelco Discovery C18 (250 mm × 10 mm, 5 µm, 300 Å) column with the same linear gradient as LC-ESI-MS at a flow rate of 4.7 mL/min. Detection was carried out at λ ) 214 nm. Mass Spectroscopy. Positive ion electrospray ionization mass spectrometric (ESI-MS) analyses of Ac-IL-1βSOC4-Ac4-TT-NH2 (1), Ac-IL-1β-SOC4-Sm4-TT-NH2 (2), and Ac-IL-1β-SOC4-[Ac2-(La/SSB)2]-TT-NH2 (3) were performed on a Micromass Platform LC-MS. Capillary and cone voltages were set to 3 kV and 45 V, respectively. Samples were dissolved in water/acetonitrile/trifluoroacetic acid (1/1/0.05 V/V/V). Circular Dichroism. CD spectra were recorded at 25°C on a Jasco J-710 spectrophotometer using 0.1 cm path-length quartz cell and peptide concentration 10-4 M. Experiments were performed in TFE/H2O mixtures (from 0 to 100%) and in SDS (concentrations ranged from 5 to 15 mM). Spectra were obtained with a 2 nm bandwidth, a scan speed of 4 nm min-1 and a time constant of 8 s. The signal-to-noise ratio was improved by accumulating four scans. To eliminate errors from instrumental drift, the baseline in air was recorded and subtracted after each scan. All CD spectra are reported in terms of ellipticity units per mole of peptide residue ([Θ]R in deg cm2 dmol-1). The percentage helical content was estimated on the basis of the [Θ]222 nm values, in different environments, as described by Chen et al. (30) and using the CD deconvolution (CDNN program) described by Bohn et al. (31). Two parameters R1 and R2 described by Rozek et al. (32) were also used for analyzing the CD spectra. R1 is the ratio of the intensity of the maximum between 190 and 195 nm and the intensity of the minimum between 200 and 210 nm. R2 is the intensity of the minimum near 222 nm and the intensity of the minimum between 200 and 210 nm. Rabbit Immunizations. New Zealand white rabbits were immunized, two for each peptide-conjugate, subcutaneously following three different protocols. Protocol A: 1 mg of each conjugate in 500 µL of H2O emulsified in 500 µL of CFA was injected on day 1, followed by boostings with 0.5 mg/500 µL H2O emulsified in 500 µL of IFA with two weeks intervals. Animals were bled after two successive immunizations on day 21, after three injections on day 43, and after five injections on day 86. Protocol B: 1 mg of each conjugate in 500 µL of H2O emulsified in 500 µL of IFA was injected following the previous schedule. Animals were also bled after the sixth injection on days 108. Protocol C: 1 mg of each conjugate in 1 mL of H2O was injected following the schedule of protocol A adding neither CFA nor IFA. Animals were also bled after the sixth injection on days 108. Mice Immunization. Mice (Balb/c) were immunized, three for each experiment, subcutaneously following two different protocols.

Carrier Immunostimulatory Effect

Protocol A: 50 µg of the conjugate (3) in 100 µL of H2O emulsified in 100 µL of CFA was injected on day 1, followed by boostings with the same quantities of immunogen emulsified in 100 µL of IFA with two weeks intervals. Animals were bled after two successive immunizations. Protocol B: 50 µg of immunogen in 200 µL of H2O was injected following the previous schedule without adding CFA/IFA. ELISA for Antisera. Ninety-six-well polystyrene plates (Nunc Immuno Plates, Denmark) were coated overnight at 4 °C with 10 µg/mL of each peptide-conjugate including the Ac-IL-1β-SOC4-Ac4-TT-NH2 carrier in carbonate buffer, pH 9.6 (100 µL/well). After washings with PBS/pH 7.2, the nonspecific binding sites were blocked with 3% skim milk in PBS and the plates were incubated at room temperature for 2 h. Serum samples from preimmunized and postimmunized animals at dilutions, with blocking buffer, ranging from 1:100 to 1:50.000, were added, and the plates were incubated at 4 °C overnight. After extensive washings with PBS, the wells were incubated with anti rabbit anti-human IgG or goat antimouse IgG conjugated to peroxidase (Jackson Immunoresearch) at 37 °C for 1.5 h. Finally the plates were washed, 50 µL of substrate solution of TMB (1 mg/1 mL) and 50 µL of H2O2 (1 mL/1 mLH2O) were added to each well, and the absorbance was measured at 450 nm. RESULTS

Design and Synthesis of an Innovative Multifunctional Carrier Bearing B and T Cell Epitopes as Potential Immunogenic Conjugates. A new carrier combining (i) the tetrameric repeating unit (Lys-AibGly)4, SOC4, for anchoring B/T cell epitopes from the LysNH2 groups to induce specific antibodies, (ii) the IL1β(163-171) fragment to improve the quality and prolong the immune response, and (iii) the TT(593-599) segment as universal immunogen, was constructed according to the principles of the step by step solid-phase peptide synthesis, following an othogonal system of N-protecting groups (Boc-, Fmoc-, and Alloc-). This approach allowed the formulation of reconstituted potent immunogenic conjugates in good yield and high purity as confirmed by ESI-MS and the corresponding LC. Molecular weights of the reported preparations ranged from 5940 to 7620, approaching the molecular mass of small proteins. Conformational Study by CD. The CD spectra of the carrier in TFE/H2O mixtures (10, 30, and 50%) showed a positive band at 192 nm and two negative bands at 208 and 222 nm typical of helical structure, which reaches maximum ellipticity at 100% TFE (Figure 3). It is presumed that trifluoroethanol favors the formation of a more ordered conformation by lowering the dielectric constant of the dissolving medium. The effect of SDS, a membrane mimetic, on the conformation of the carrier was also studied. In phosphate buffer, pH 6, the CD spectrum exhibits weak helical characteristics. Upon addition of SDS, at concentration 5 mM, below the critical micelle concentration (CMC 8 mM) the typical characteristic bands of helical structure appeared, indicating that upon interaction with SDS even below CMC the helical character of the carrier is enhanced (33). The ellipticity remained practically unchanged at 10 and 15 mM SDS, suggesting that the micelle microenvironment at 5 mM is sufficient to induce maximum helical conformation to the carrier. The percentage helical content of the carrier in phosphate buffers, 5 mM SDS and 100% TFE, calculated on

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Figure 3. CD spectra of the Ac-IL-1β-SOC4-Ac4-TT-NH2 (1) carrier in TFE/H2O mixtures 10, 30, 50, and 100% TFE.

the basis of the [Θ]222 value (30) and the CD deconvolution (31) was estimated to 6, 20, and 40%, respectively. Moreover, R1 parameter is close to -2 and R2 approximates 1, confirming the helicoid character of the carrier (32). It seems likely that the helicoid features of SOC4 (1014, 34, 35) are conserved even after its amino and carboxy terminus elongation. One may assume that the persistence of the ordered secondary conformation of the AcIL-1β-SOC4-Ac4-TT-NH2 carrier could optimize the B/T cell epitope presentation and help to the formation of functional reconstituted immunogenic conjugates. Serological Study. Comparison of the Humoral Response in Rabbits Immunized with Three Different Approaches. Sera were collected before the initial immunization, as well as after two subsequent boosts and were tested against the immunizing conjugate. The reactivity of rabbit anti-sera to the Ac-IL-1βSOC4-Sm4-TT-NH2 conjugate (2) following the CFA/IFA procedure increased gradually and remained at high levels after the third immunizatiuon. Dilution curves of sera in anti-Ac-IL-1β-SOC4-Sm4-TT-NH2 ELISA are shown in Figure 4A. Rabbits immunized with the Ac-IL-1βSOC4-Sm4-TT-NH2 conjugate (2) and IFA raised specific antibodies recognizing the priming immunogen after the sixth boosting. Sera samples displayed high reactivity even at 1:50000 dilution (Figure 4B). Antibody specific response in rabbits immunized with Ac-IL-1β-SOC4-Sm4TT-NH2 (2) alone, without administering neither CFA nor IFA was detected after the sixth injection and sera reactivity, as shown in anti-Ac-IL-1β-SOC4-Sm4-TT-NH2 ELISA tests, remained high even at 1:50000 dilution (Figure 4C). Animals were immunized with Ac-IL-1β-SOC4-[Ac2(La/SSB)2]-TT-NH2 congugate (3) following the previous protocols, and sera were tested for the induction of antiAc-IL-1β-SOC4-[Ac2-(La/SSB)2]-TT-NH2 antibodies in ELISA tests. The pattern of the sera reactivity was comparable with that observed when conjugate 2 was injected and the anti-conjugate 3 sera reactivity remained high (1:50000 dilution) even when conjugate 3 was injected alone without the assistance of adjuvant (Figure 5). Usage of adjuvant is essential to induce strong and prolonged antibody responses although their function is not fully understood. Adjuvants incorporate two components. One is a substance designed to form a deposit protecting the antigen from rapid catabolism, and the other is a substance that stimulates the immune response nonspecifically by activating the antigen-processing cells

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Figure 4. Binding of antisera to the Ac-IL-1β-SOC4-Sm4-TTNH2 (2) conjugate. Antisera raised in rabbits immunized with CFA/IFA (A), IFA (B), and the peptide conjugate alone (C). Data shown as means of three independent experiments carried out in triplicate using a pool of the two rabbit sera that gave similar results when used independently. Control sera: preimmune.

directly. Freund’s adjuvant, which is the most commonly used adjuvant for research work, is a water-in-oil emulsion prepared with nometabolizable oils that contains heat-killed tuberculosis mycobacteria. The principal disadvantage of Freund’s adjuvant is that it can cause a local inflammatory reaction at the site of injection and invoke very aggressive and persistent granulomas. To avoid these side effects, incomplete Freund’s adjuvant, among others, has been proposed although severe skin toxicities have been reported in some cases (3, 4, 36, 37). The profile of the antibody production when immunogens Ac-IL-1β-SOC4-Sm4-TT-NH2 and Ac-IL-1β-SOC4[Ac2-(La/SSB)2]-TT-NH2 were injected alone leads to the assumption that incorporation of Aib, an enzymatic nondegradable residue, contributes to the slow release of epitopes thus resembling the proposed action of the nometabolizable oily constituents of IFA. On the other hand, the inclusion into the carrier of the IL-1β(163-

Krikorian et al.

Figure 5. Binding of antisera to the Ac-IL-1β-SOC4-[Ac2,(La/ SSB)2]-TT-NH2 conjugate (3). Antisera raised in rabbits immunized with CFA/IFA (A), IFA (B), and the peptide conjugate alone (C). Data shown as means of three independent experiments carried out in triplicate using a pool of the two rabbit sera that gave similar results when used independently. Control sera: preimmune

171) fragment, known to reproduce the immunostimulatory and adjuvant functions of the whole IL-1β but not its numerous side effects (15, 16), assisted the generation of an enhanced and sustained immune response, as that of CFA, which is also site specific, as the produced antibodies, so far presented, recognize the priming immunogens. Specificity of Antibody Recognition. To further assess the specificity of the generated antibodies when the constructed immunogens were injected to animals, the obtained antisera were tested against the carrier AcIL-1β-SOC4-Ac4-TT-NH2 (1) and SOC4, Ac-[Lys(Ac)-AibGly]4, without bearing any peptide epitope. Antibodies raised against peptide-conjugate 2 are highly recognized by the priming peptide, but their recognition from the carrier, which incorporates the IL-1β and TT fragments, and SOC4, when administered without adjuvant is negligible (Figure 6A). Similarly, antibodies raised against peptide-conjugate 3 without adjuvant exhibit negligible reactivity against the carrier compared to their high

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Figure 6. Comparison of the antisera reactivity of rabbits immunized with conjugate Ac-IL-1β-SOC4-Sm4-TT-NH2 (2) against 2, the Ac-IL-1β-SOC4-Ac4-TT-NH2 carrier (1), and the SOC4 carrier (A). Comparison of the antisera reactivity of rabbits immunized with conjugate Ac-IL-1β-SOC4-[Ac2-(La/SSB)2]-TTNH2 (3) against (3) the Ac-IL-1β-SOC4-Ac4-TT-NH2 carrier (1), and the SOC4 carrier (B). Peptide conjugates 2 and 3 were administered with CFA/IFA, IFA, and without adjuvant as indicated. Sera dilution: 1/800. Figure 8. Binding of antisera to the Ac-IL-1β-SOC4-[Ac2-(La/ SSB)2]-TT-NH2 (3) conjugate at 1/500 dilution. Antisera raised in mice immunized with CFA/IFA (A) and the peptide conjugate alone without adjuvant (B). Data shown as means of three independent experiments carried out in triplicate using a pool of three mice sera that gave similar results when used independently. Control sera: preimmune (C).

Figure 7. Comparison of the antisera reactivity of rabbits immunized (without adjuvant) with conjugate Ac-IL-1β-SOC4[Ac2-(La/SSB)2]-TT-NH2 (3) alone (without adjuvant) against 3, recombinant La/SSB (Rec La), N-terminal recombinant La/SSB (N-Rec-La), and C-terminal recombinant La/SSB (C-Rec-La).

recognition by the priming peptide (Figure 6B). These findings indicate that the reported new immunogenic peptide-conjugates induce (without the assistance of adjuvant) a specific enhanced immunostimulatory response without producing nonspecific antibodies recognizing SOC4 (23, 38) or the elongated by IL-1β and TT new carrier. Rabbit antisera induced after immunization with peptide-conjugate 3 alone, without adjuvant, displayed significant reactivity (∼60%) against recombinant La/SSB (39) and ∼40% reactivity against the C-terminus and N-terminus of the recombinant La/SSB (39) compared to the anti-peptide-conjugate 3 antibody recognition (Figure 7). One may assume that immunizations using the conjugates presented in this study, without the addition of an adjuvant, results in polyclonal antibody response against the cognate protein or parts of it, as it was already mentioned when the conventional immunization protocol (CFA/IFA) was followed, suggesting the activation of antigen-specific B cells (23, 41). In an attempt to validate the efficacy of our peptideconjugates in species other than rabbits, immunizations were performed in mice, applying the peptide-conjugate

3 following the CFA/IFA protocol and without the addition of adjuvant (Figure 8). Although rather lower titers of antibodies were produced when conjugate 3 was administered alone, the immunostimulatory response was specific for the priming peptide-conjugate, indicating the utility of the promiscuous T cell epitope, TT(593599), in providing a broader spectrum of immunogenicity (3, 5, 6). DISCUSSION

A new multivalent carrier combining B and T cell epitopes, a built-in adjuvant and a “promiscuous” T cell epitope as costimulatory molecules to formulate potential immunogenic conjugates is presented. Following the stepby-step solid-phase peptide synthesis principles and by applying orthogonal side-chain protections, it was feasible to synthesize two peptide-conjugates, as mimics of the Sm and La/SSB proteins, with accurate and unambiguous composition and structure without altering the initial properties of the conjugated epitopes. Moreover, is very probable that the helicoid features of the presented multifunctional carrier, as revealed by CD measurements, contribute to the conformation-dependent recognition of B cell epitopes of Sm and La/SSB proteins to elicit specific antibodies. In particular, the IL-1β(163171) fragment was covalently linked to the N-terminus of the tetrameric Sequential Oligopeptide Carrier, SOC4, as immunostimulator, while the TT(593-599) sequence was attached to the carboxy-terminus of SOC4 as universal immunogen. The reconstituted Sm and La/SSB immunogenic conjugates 2 and 3 respectively when injected in animals,

818 Bioconjugate Chem., Vol. 16, No. 4, 2005

without the assistance of CFA/IFA, induced high titers of specific antibodies recognizing the priming peptideconstructs. The antibody reactivity remained high even at high sera dilution (1:50000), indicative of the potency and the good quality of the immune response. It seems likely that the inclusion of IL-1β(163-171) within the synthesized reconstituted immunogenic congugates may substitute the immunostimulatory activity of Freund’s adjuvant, while incorporation of Aib, an enzymatic nondegradable residue, in the repeating tripeptide unit of SOC4 may replace the function of the nonmetabolizable oily constituents of the incomplete Freund’s adjuvant. Confirmation of the specificity of the generated antibodies, when the constructed immunogens are injected without the assistance of adjuvant, is further assessed from the fact that they do not recognize the Ac-IL-1βSOC4-Ac4-TT-NH2 carrier, which comprises the IL-1β, and the TT fragments and SOC4. On the basis of these findings, it is presumed that the immune response is antigen driven, originated from the reported conjugates. It is of particular interest to underline the defined intramolecular spreading of the antibody response when epitope La/SSB(289-308) is injected in the form of AcIL-1β-SOC4-[Ac2-(La/SSB)2]-TT-NH2 without the assistance of Freund’s adjuvant. The observed immunogenicity of construct 3 when injected in rabbits and mice implies that the inclusion of TT(593-599) in the reported carrier could help to overcome genetic restrictions of the T cell “help” to B cells. Our study provides the conceptual and experimental framework to formulate multifunctional immunogens as human vaccine candidates without the adverse effects derived from the usage of adjuvants. ACKNOWLEDGMENT

These studies were supported by grants from the Greek General Secretariat of Research and Technology (GSRT). Supporting Information Available: Schematic representation of the synthesis of the Ac-IL-1β-SOC4-Ac4-TTNH2 carrier (1) and Ac-IL-1β-SOC4-[Ac2-(La/SSB)2]-TTNH2 conjugate (3). LC and ESI-MS of the Ac-IL-1β-SOC4Ac4-TT-NH2 carrier (1), and Ac-IL-1β-SOC4-Sm4-TT-NH2 (2) and Ac-IL-1β-SOC4-[Ac2-(La/SSB)2]-TT-NH2 (3) conjugates. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Kaumaya, P. T., Berndt, K. D., Heidorn, D. B., Trewhella, J., Kezdy, F. J., and Goldberg, E. (1990) Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology. Biochemistry 29, 13-23. (2) Kobs-Conrad, S., Lee, H., DiGeorge, A. M., and Kaumaya, P. T. (1993) Engineered topographic determinants with alpha beta, beta alpha beta, and beta alpha beta alpha topologies show high affinity binding to native protein antigen (lactate dehydrogenase-C4). J. Biol. Chem. 268, 25285-25295. (3) Sundaram, R., Dakappagari, N. K., and Kaumaya, P. T. (2002) Synthetic peptides as cancer vaccines. Biopolymers 66, 200-216. (4) Reid, C. D., Stackpoole, A., Meager, A., and Tikerpae, J. (1992) Interactions of tumor necrosis factor with granulocytemacrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow. J. Immunol. 149, 2681-2688. (5) Kaumaya, P. T., Kobs-Conrad, S. Seo, Y. H., Lee, H., VanBuskirk, A. M., Feng, N., Sheridan, J., and F. Stevens, V. (1993) Peptide vaccines incorporating a ′promiscuous' T-cell

Krikorian et al. epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity. J. Mol. Recognit. 6, 81-94. (6) Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S., Corradin, G., and Lanzavecchia, A. (1989) Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur. J. Immunol. 19, 2237-2242. (7) Metzger J., Wiesmuller K. H., Schaude, R., Bessler, W. G., and Jung, G. (1991) Synthesis of novel immunologically active tripalmitoyl-S-glycerylcysteinyl lipopeptides as useful intermediates for immunogen preparations. Int. J. Pept. Protein Res. 37, 46-57. (8) Postnett, D. N., McGrath, H., and Tam, J. P. (1988) A novel method for producing anti-peptide antibodies: production of site site-specific antibodies to the T-cell antigen receptor β-chain. J. Biol. Chem. 263, 1719-1725. (9) Mutter, M., Dumy, P., Garrouste, P., Lehmann, C., Mathieu, M., Peggion, C., Peluso, S., Razaname, A., and Tuchscherer, G. (1996) Template assembled synthetic proteins (TASP) as functional mimetics of proteins. Angew. Chem., Int. Ed. Engl. 35, 1482-1485. (10) Tsikaris, V., Sakarellos, C., Cung, M. T., Marraud, M., and Sakarellos-Daitsiotis, M. (1996) Concept and design of a new class of sequential oligopeptides carrier (SOC) for covalent attachment of multiple antigenic peptides. Biopolymers 38, 291-293. (11) Tsikaris, V., Sakarellos, C., Sakarellos-Daitsiotis, M., Orlewski, P., Marraud, M., Cung, M. T., Vatzaki, E., and Tzartos, S. (1996) Construction and application of a new class of sequential oligopeptide carries (SOCn) for multiple anchoring of antigenic peptides-application to the acetylcholine receptor (AChR) main immunogenic region. Int. J. Biol. Macromol. 19, 195-205. (12) Sakarellos-Daitsiotis, M., Tsikaris, V., Sakarellos, C., Vlachoyiannopoulos, P. G., Tzioufas, A. G., and Moutsopoulos, H. M. (2000) A new helicoid-type sequential oligopeptide carrier (SOCn) for developing potent antigens and immunogens. Vaccine 18, 302-310. (13) Sakarellos-Daitsiotis, M., Tsikaris, V., Vlachoyiannopoulos, P. G., Tzioufas, A. G., Moutsopoulos, H. M., and Sakarellos, C. (1999) Peptide Carriers: A helicoid-type sequential oligopeptide carrier (SOCn) for multiple anchoring of antigenic/ Immunogenic Peptides. Methods, A Companion to methods in Enzymology 19, 133-141. (14) Keramisanou, D., Tsikaris, V., Sakarellos-Daitsiotis, M., Sakarellos, C., Mikhailova, A. A., and Strelkov, L. A. (2002) Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOCn): synthesis, conformation and studies in human leukemia cells. J. Pept. Res. 59, 1-8. (15) Tagliabue, A., and Boraschi, D. (1993) Cytokines as vaccine adjuvants: Interleukin 1 and its synthetic peptide 163-171. Vaccine 11, 594-595. (16) Boraschi, D., and Tagliabue, A. (1999) Interleukin-1 and Interleukin-1 fragments as vaccine adjuvants. Methods 19, 108-113. (17) Ho, P. C., Mutch, D. A., Winkel, K. D., Saul, A. J., Jones, G. L., Doran, T. J., and Rzepczyk, C. M. (1990) Identification of two promiscuous T-cell epitopes from tetanus toxin. Eur. J. Immunol. 20, 477-483. (18) James, J. A., and Harley, J. B. (1992) Linear epitope mapping of an Sm B′/B polypeptide. J. Immunol. 148, 20742079. (19) Tan, E. M., Cohen, A. S., Fries, J. M., Mashi, A. T., McShane, D. J., Rothfield, N. F., Schallez, J. G., Talal, N., and Winchester, R. J. (1982) The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25, 1271-1277. (20) Petrovas, C. J., Vlachoyiannopoulos, P. G., Tzioufas, A. G., Alexopoulos, C., Tsikaris, V., Sakarellos-Daitsiotis, M., Sakarellos, C., and Moutsopoulos, H. M. (1998) A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies. J. Immunol. Methods 220, 59-68. (21) Fatenejad, S., Mamula, M., and Craft, J. (1993) Role of intermolecular/intrastructural B- and T- cell determinants

Bioconjugate Chem., Vol. 16, No. 4, 2005 819

Carrier Immunostimulatory Effect is the diversification of autoantibodies to ribonucleoprotein particles. Proc. Natl. Acad. Sci. U.S.A. 90, 12010-12014. (22) Tzioufas, A. G., Yiannaki, E., Sakarellos-Daitsiotis, M., Routsias, J. G., Sakarellos, C., and Moutsopoulos, H. M. (1997) Fine specificity of autoantibodies to La/SSB. Epitope mapping molecular mimicry and clinical applications. Clin. Exp. Immunol. 108, 191-198. (23) Yiannaki, E., Vlachoyiannopoulos, P. G., Manousakis, M. N., Sakarellos, C., Sakarellos-Daitsiotis, M., Moutsopoulos, H. M., and Tzioufas, A. G. (2000) Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La/SSB autoantigen. Clin. Exp. Immunol. 121, 551-556. (24) Stewart, J. M., and Young, J. D. Principles of Peptide Synthesis, Second ed. Pierce Chemical Co, Rockford IL, 1984. (25) Bodansky, M. Principles of Peptide Synthesis, Second ed. Springer: Berlin, 1993. (26) Bodansky, M., and Bodansky, A. The practice of Peptide Synthesis, Second ed. Springer: Berlin, 1994. (27) Kaiser, E., Bossinger, C. D., Colescott, R. L., and Olsen, D. B., (1980) Color test for terminal prolyl residues in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595598. (28) Dangles, O., Guibe, F., Balavoine, G., Lavielle, S., and Marquet, A. (1987) Selective cleavage of the allyl and allyloxycarbonyl groups through palladium-catalyzed hydrostannolysis with tributyltin hydride. Application to the selective protection-deprotection of amino acid derivatives and in peptide synthesis. J. Org. Chem. 52, 4984-4993. (29) Loffet, A., and Zhang, H. X. (1993) Allyl-based groups for side-chain protection of amino acids. Int. J. Peptide, Protein Res. 42, 346-351. (30) Chen, Y. H., Yang, J. T., and Martinez, H. M. (1972) Determination of the secondary structures of proteins by circular dishroism and optical rotatory dispersion. Biochemistry 11, 4120-4131. (31) Bohm, G., Muhr, R., and Jaenicke, R. (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng. 5, 191-195. (32) Rozek, A., Buchko, G. W., and Cushley, R. J. (1995) Conformation of two peptides corresponding to human apolipoprotein C-I residues 7-24 and 35-53 in the presence of

sodium dodecyl sulfate by CD and NMR spectroscopy. Biochemistry 34, 7401-7408. (33) Fasman, G. D. (1996) Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York. (34) Alexopoulos, Ch., Tsikaris, V., Rizou, C., SakarellosDaitsiotis, M., Sakarellos, C., Cung, M. T., Marraud, M., Vlachoyiannopoulos, P. G., and Moutsopoulos, H. M. (2000) The position of the Lys-NH2 grafted antigens along the sequential Oligopeptide carrier, Ac-(Aib-Lys-Aib-Gly)n (SOCnII), influences the antibody recognition: application to the Sm main autoimmune epitope. Biopolymers 54, 1-10. (35) Sakarellos-Daitsiotis, M., Alexopoulos, Ch., and Sakarellos, C. (2004) Sequential oligopeptide carriers, SOCn, as scaffolds for the reconstitution of antigenic proteins: applications in solid-phase immunoassays. J. Pharm. Anal. 34, 761-769. (36) Gupta, R. K., Relyveld, E. H., Lindblad, E. B., Bizzini, B., Ben-Efraim, S., and Gupta, C. K. (1993) Adjuvants-a balance between toxicity and adjuvanticity. Vaccine 11, 293-306. (37) Gupta, R. K., and Siber, G. R. (1995) Adjuvant for human vaccines-current status problems and future prospects. Vaccine 13, 1263-1276. (38) Vlachoyiannopoulos, P. G., Petrovas, C., Tzioufas, A. G., Alexopoulos, Ch., Tsikaris, V., Guialis, A., Nakopoulos, L., Sakarellos-Daitsiotis, M., Sakarellos, C., Davaris, P., and Moutsopoulos, H. M. (2000) No evidence of epitope spreading after immunization with the magor Sm epitope P-P-G-MR-P-P anchored to Sequential Oligopeptide Carriers (SOCs). J. Autoimm. 14, 53-61. (39) Yiannaki, E. E., Tzioufas, A. G., Bachmann, M., Hantoumi, J., Tsikaris, V., Sakarellos-Daitsiotis, M., Sakarellos, C., and Moutsopoulos, H. M. (1998) The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB.; specificity, sensitivity and comparison of methods. Clin. Exp. Immunol. 112, 152-158. (40) Mamula, M. J., and Janeway, C. A. Jr. (1993) Do B cells drive the diversification of immune responses? Immunol. Today 14, 151-152. (41) Craft, J., and Fatenejad, S. (1997) Self-antigens and epitope spreading in systemic autoimmunity. Arthritis Rheum. 10, 1374-1382.

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