Immunogenic Peptides

METHODS 19, 133–141 (1999) Article ID meth.1999.0838, available online at http://www.idealibrary.com on

Peptide Carriers: A Helicoid-Type Sequential Oligopeptide Carrier (SOC n) for Multiple Anchoring of Antigenic/Immunogenic Peptides Maria Sakarellos-Daitsiotis,* Vassilios Tsikaris,* Panayiotis G. Vlachoyiannopoulos,† Athanassios G. Tzioufas,† Haralampos M. Moutsopoulos,† and Constantinos Sakarellos* ,1 *Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, G-45110 Ioannina, Greece; and †Department of Pathophysiology, Medical School, National University of Athens, Athens, Greece

A new peptide carrier with three-dimensional predetermined structural motif has been constructed by the repetitive Lys–Aib– Gly moiety. The sequential oligopeptide carrier (SOC n), (Lys–Aib– Gly) n, adopts a distorted 3 10-helical conformation and the Lys– N eH 2 anchoring groups exhibit defined spatial orientations. Conformational analysis of the SOC n conjugates showed that the coupled peptides retain their initial “active” structure, while prevalence of one conformer was also observed. It is concluded that the beneficial structural elements of SOC n induce a favorable arrangement of the conjugated peptides, so that potent antigens and immunogens are generated. © 1999 Academic Press

In recent years the development of improved methods of peptide synthesis, together with the revolution in molecular technology, has allowed the replacement of crude antigenic extracts with highly pure, recombinant and/or synthetic antigens. Short synthetic peptides can mimic linear and, to a certain extent, conformational epitopes and they can be used in solid-phase assays instead of complex antigens that are often difficult to purify and prepare in large quantities. The availability of peptides has stimulated a number of investigations for the detection and, in some cases, for the discrimination of antibodies in patient sera. It is also possible that peptides, rather than whole isolated proteins, resemble more closely short regions accessible at the surface of macromolecular complexes, which 1 To whom correspondence should be addressed. Fax: 130 (651) 45203. E-mail: [email protected]

1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

may be involved as immunogens in the autoimmune response. Studies with short protein fragments may help to propose new therapeutic strategies by designing, for example, modified peptides able to affect the immune system by interfering at different levels [e.g., antigen presentation by the major histocompatibility complex (MHC) molecules] or by using a drug targeting approach based on antipeptide antibodies (1–3). To obtain potent antigens or immunogens, however, synthetic peptides (epitopes or antigenic determinants) must be conjugated to a carrier, for example, bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin. Although this approach has been used successfully in many cases to produce antibodies, some crucial disadvantages may prevent the application of such conjugates in human vaccines: (i) immune response to the carrier, (ii) ambiguous composition and structure, (iii) alteration of the biologically “active” conformation of the coupled antigenic determinant, and (iv) the peptide antigen, which represents only a small portion of the total antigen– carrier conjugate, may result in a minor fraction of specific antibodies (4 – 6).

DESCRIPTION OF METHODS Artificial Peptide Carriers Since antibodies are one of the first lines of defense against invading organisms, synthetic peptides offer a powerful approach to a new generation of vaccines. An ideal peptide vaccine should incorporate the following elements: (i) B-cell epitopes that mimic the three133

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FIG. 1. llustration of the sequential oligopeptide carrier (SOC n) and the anchored antigenic/immunogenic peptides A: [Ala 76]–MIR, gp63–SRYD, PPGMRPP, and La/SSB 349 –364.

dimensional conformation of the antigen, (ii) T-cell epitopes resulting from the “processed” fragment of a protein antigen, which must selectively bind to the class II MHC, (iii) both B- and T-cell-recognizing determinants, on the same molecule (artificial carrier),

(iv) the artificial carrier should provide the binding site between class II MHC and T-cell receptor, (v) “promiscuous” T-cell epitopes, which should bind a broad spectrum of isotypic and allotypic forms of human MHC, and (vi) appropriate adjuvants (other than bacterial components) with immunostimulating activity without side effects (7). Obviously, accomplishment of the above-mentioned prerequisites requires the design and construction of a carrier, encompassing multiple functionalities, other than the conventional protein carriers. Branching Lysine Core Matrix Multiple antigenic peptides (MAPs) introduced by Tam have demonstrated several advantages in producing antipeptide antibodies. In the MAP system, multiple copies of the peptide antigen are attached to a

FIG. 2. Steps in the synthesis of the sequential oligopeptide carrier (SOC n) (A) and the SOC n conjugates (B).

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ANTIGENIC/IMMUNOGENIC PEPTIDE CARRIERS TABLE 1 Amide Chemical Shifts d, Temperature Coefficient Values a (Dd/DT), and 3J Na, 3J Na9 Coupling Constants a of Ac(OLysOAibOGly) n P Ac a

Lys 1

SOC n Ac(LysOAibOGly) 4 P Ac Ac(LysOAibOGly) 5 P Ac

d Dd /DT 3 J Na 3 J Na9 d Dd /DT 3 J Na 3 J Na9

Aib 2

Gly 3

Lys 4

Aib 5

Gly 6

Lys 7

Aib 8

Gly 9

Lys 10

Aib 11

Gly 12

Lys 13

Aib 14

Gly 15

8.13 8.50 8.14 7.91 b 8.14 d 8.17 f 7.91 i 8.67 c 8.36 e 7.46 g 8.00 h 7.12 j 24.1 26.2 23.8 20.3 24.1 23.2 21.6 27.0 23.5 21.1 24.6 21.8 6.7 5.9 6.6 5.9 7.4 6.6 7.5 4.7 5.7 5.6 5.3 4.3 b d f i c 8.09 8.57 8.15 7.73 8.02 7.99 7.92 8.14 8.15 7.94 8.62 8.30 e 7.45 g 7.96 h 23.6 27.2 24.3 20.7 24.1 23.2 10.2 25.0 23.7 22.8 27.4 23.2 20.8 24.2 6.7 5.7 7.3 5.9 5.9 6.4 7.6 6.4 7.4 5.3 7.7 5.4 5.4

7.10 j 21.7 4.7 3.9

d (ppm) referenced to (CH 3) 4 Si; Dd/DT (10 23 ppm K 21); 3J (Hz). n 5 4,5. In DMSO-d 6 (0.5– 0.7 3 10 22 M) at 300 K, originally obtained from aqueous solutions at pH 5. b–j Similarities of chemical shifts for the specific amide protons of both compounds (b-b, c-c, d-d, e-e, f-f, g-g, h-h, i-i, j-j). a

trifunctionalized lysine scaffolding, which forms a core multidendritic matrix to generate a macromolecular peptidic immunogen (8, 9). However, some limitations of the MAP system in eliciting high titers of antipeptide antibodies recognizing the native protein, as well as in the development of efficient antigens in enzymelinked immunosorbent assay (ELISA) tests, have recently been reported (10). Also, difficulties in the synthetic procedure have been reported, attributed to the formation of incomplete peptide sequences. It is very probable that these drawbacks in the use of MAPs result from steric hindrances due to the close proximity of the antigenic peptides on the lysine tree. Various modified MAP systems have been appeared, for example: (i) a more soluble lysine core matrix incorporating two N-(S-acetylmercaptoacetyl)glutamyl residues for each lysine (11, 12) and (ii) a lysine tree bearing eight cysteine residues, presenting eight SH groups for reacting with an activated antigen. Much effort has also been expended in using unprotected peptides for ligation to the lysine scaffold (13, 14).

Lipopeptides consisting of the synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy-(2RS)-propyl]-[R]cysteine and peptides from the N terminus of a bacterial lipoprotein have been proven to be macrophage and B-cell activators in vaccine preparations (15). Also noteworthy is the template assembled synthetic protein (TASP) concept, in de novo protein design for the construction of proteinlike molecules, exhibiting tailormade functional properties. Attempts have been made to prepare topological templates with catalytic and immunological properties (16, 17). Sequential Oligopeptide Carriers (SOC n) One of the major guidelines in designing this new class of carriers was to model constructions with predetermined three-dimensional structure, so that the attached antigenic peptides would attain a defined spatial orientation. Our scaffold, formed by the repetitive Lys–Aib–Gly moiety, incorporates lysine for anchoring of the antigen, the a-aminoisobutyric residue for induc-

TABLE 2 Dimensions and Hydrogen Bonds Occurrence (.10%) in SOC 4 a

a b c

Donor

Acceptor

Dd/DT b

Interaction

Distance c

Occurrence (%)

Lys 4–NH Aib 5–NH Lys 7–NH Gly 9–NH Lys 10–NH Lys 10–NH Gly 12–NH

Lys 1–CO Gly 3–CO Lys 4–CO Gly 6–CO Gly 6–CO Lys 7–CO Gly 9–CO

20.3 24.1 21.6 23.5

i133i i123i i133i i133i i143i i133i i133i

2.19 2.18 2.24 2.33 2.25 2.19 2.17

72 65 87 11 10 90 78

21.1 21.8

In vacuo-restrained MD simulation during last 20 ps. Temperature coefficient values (in 10 23 ppm K 21) for the NH proton resonance. N–HzzzO distance between the nonhydrogen atoms in Å.

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The following selected peptide antigens were chosen for applying the SOC n.

FIG. 3. Minimized average structure of the sequential oligopeptide carrier (SOC 4) from the in vacuo-restrained MD simulation during the last 20 ps. Comparison to the ribbon representing a canonical 3 10-helix of the SOC 4 backbone. Reprinted from Int. J. Biol. Macromol. 19, V. Tsikaris, C. Sakarellos, M. Sakarellos-Daitsiotis, P. Orlewski, M. Marraud, M. T. Cung, E. Vatzaki, and S. Tzartos, 195– 205, Copyright 1996, with permission from Elsevier Science.

ing a helicoid structure of the peptide backbone, and glycine for its small stereochemical volume. The sequential oligopeptide carrier Ac–(Lys–Aib–Gly) n (n 5 2–7), named SOC n, is expected to adopt a regular secondary structure, thus allowing the antigenic peptides to retain their natural “active” conformation without interacting with each other or with the carrier (Fig. 1). The structure regularity of the carrier, the absence of conformational restrictions, and steric hindrances for the constructed conjugates would favor an optimal antibody recognition and generate potent immunogens (18, 19).

i. The [Ala 76] analogs of the main immunogenic region (MIR, Trp 67–Asn–Pro–Ala–Asp–Tyr–Gly–Gly– Ile–Lys 76] of the a subunit of Torpedo nicotinic acetylcholine receptor (AChR). Against this region, a67–76, is directed the majority of the anti-AChR autoantibodies from myasthenic patients, which cause loss and/or blockage of AChR function and failure of neuromuscular transmission (20, 21). ii. The Ile 250–Ala–Ser–Arg–Tyr–Asp–Gln–Leu 257 fragment (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63. This sequence efficiently inhibits parasite attachment to the macrophage receptors and the Ser–Arg–Tyr–Asp (252–255) moiety represents the putative gp63 adhesion site (22, 23). iii. The PPGMRPP sequence, found in several copies in the Sm and U1RNP autoantigens, is the main target of anti-Sm antibodies in sera of patients with systemic lupus erythematosus (SLE). This sequence has been recognized mainly by anti-Sm and, to a lesser extent, by anti-U1RNP sera (24). iv. The B-cell antigenic determinants of the La/ SSB antigen: TLHKAFKGSIFVVFDSIESA (145–164), ANNGNLQLRNKEVTWEVLEG (289 –308), VTWEVLEGEVEKEALKKI (301–318), and GSGKGKVQFQGKKTKF (349 –364). The bulk of anti-La/SSB reactivity in sera of patients with primary Sjo¨gren’s syndrome (pSS) and SLE is directed against the above sequences; previous clinical or experimental studies have shown that the anti-La/SSB response may be antigen driven and disease specific (25, 26). Steps in Synthesis of SOC n Conjugates The synthesis of the sequential oligopeptide carrier (SOC n) is carried out by a stepwise solid-phase procedure (18, 19). After completion of the synthesis to the desired length of carrier, the antigens are coupled simultaneously to the Lys–N eH 2 groups of SOC n, following the stepwise solid-phase Boc strategy (Fig. 2). The

TABLE 3 Chemical Shifts of Arg and Asp Side Chains of SRYD a Moiety in the Free and Bound State in DMSO-d 6 Peptide

DdR–C bH 2

DdR–C gH 2

DdR–C dH 2

dR–N eH

dR–N hH

DdD–C bH 2

Dd/DT 3 10 3 D-NH

Ac–SRYD–NH 2 IASRYDQL gp63–SRYD–SOC 6

0.48 0.59 0.46

0.05 0.11 0.05

0.20 0.24 0.20

10.45 10.18 10.02

6.99 7.05 7.03

0.49 0.42 0.33

20.2 10.5 0.0

N eH strong hydrogen bonded

N hH nonhydrogen bonded

Restricted mobility

Inaccessible to solvent

Restricted mobility

a

d is the chemical shift (ppm) and Dd is the chemical shift difference (ppm) for the corresponding geminal protons.

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peptide is cleaved with HF and subjected to appropriate dialysis, partition chromatography, and HPLC to gain maximum purity. The purity and identity of all conjugates are confirmed by analytical HPLC, amino acid analysis, and 1H NMR spectroscopy.

FIG. 4. 1H NMR spectra of PPGMRPP (I), PPGMRPP-NH 2 (II), and (PPGMRPP) 5-SOC 5 (III). Comparison of the NH regions. PPGMRPP (I): (A), (B), and (C) conformers. Reprinted from Letters in Peptide Science, 4, 1997, pp. 447– 454, C. Sakarellos, V. Tsikaris, E. PanouPomonis, C. Alexopoulos, M. Sakarellos-Daitsiotis, C. Petrovas, P. G. Vlachoyiannopoulos, and H. M. Moutsopoulos, Fig. 2, © 1997, with kind permission from Kluwer Academic Publishers.

FIG. 5. n 5 7.

Conformational Charaterization of SOC n The structural characterization of SOC n is carried out by 1H NMR experiments and molecular modeling (18, 19). In Table 1 are some NMR data on SOC n (n 5 4,5). Intense NOE connectivities, appearing between successive amide protons, are compatible either with a helical conformation or with a random structure. The latter has to be excluded due to the following findings: (i) the low absolute temperature coefficient values of all the Lys–NH indicate that they are involved in intramolecular interactions; (ii) the variation of the 3J Na and 3 J Na9 coupling constants for the glycines, except the C-terminal one, versus the torsional angle Gly-f indicates that Gly-f assumes (20) a defined value of about 670°; and (iii) a remarkable similarity between the amide proton chemical shifts of the same repeating residue, when increasing the carrier length, indicates that the Lys–Aib–Gly segments share a common repetitive conformation initiated from the carboxy end of SOC n. From the preceding NMR data it is concluded that the carrier adopts a rigid conformation with some regularity. The NMR data are introduced in MD calculations to refine the SOC n structure. Considering the main conformational angles of the time-averaged structure and the average interproton distances, estimated from NOE measurements, a time-averaged structure stabilized by a network of hydrogen bonds is obtained. The most frequent ones are of the i 1 3 3 i type, and effectively involve NH having the lower ab-

Monoclonal antibody binding to [Ala 76]MIR (a) and (WNPADYGGIA)–SOC n: (b) n 5 2, (c) n 5 3, (d) n 5 4, (e) n 5 5, (f) n 5 6, (g)

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solute temperature coefficients. The time-averaged structure of SOC n is a distorted 3 10-helix with somewhat curved axis and rms deviation between every atom pair of a canonical 3 10-helix and a SOC n less than 1.4 Å (Table 2, Fig. 3). Conformational Characteristics of SOC n Conjugates 1. Preservation of Original “Active” Conformation of Antigenic Peptide after Anchorage to SOC n The one-dimensional 1H NMR spectrum of [Ala 76]MIR–SOC 4 shows a single set of chemical shifts for the same protons of the four [Ala 76]MIR peptides bound to SOC 4. For example, the aromatic protons of the four Tyr and Trp residues appear as sharp superimposed signals. The same also holds true for the HOHAHA spectrum. These observations lead to the suggestion that the [Ala 76]MIR peptides, anchored to SOC n, are found under the same average magnetic environment. With the combined use of COSY, HOHAHA, and NOESY experiments all protons of the [Ala 76]MIR peptides bound to SOC n are assigned. The very small perturbations between the chemical shifts of the [Ala 76]MIR free and bound to SOC n, as well as the common NOE pattern, indicate that the antigenic peptides retain the same structure in both states with-

out interacting with each other (18, 19). One may note the occurrence of a b-folded Asn–Pro–Ala–Asp sequence present in the free and bound state, which was shown to be a prerequisite for optimal antibody recognition (20, 21). The structure of the gp63–SRYD–SOC 6 conjugate is analyzed in a similar way (COSY, HOHAHA, and NOESY experiments) as for [Ala 76]MIR–SOC 4. The conservative structure of the gp63–SRYD peptide, free and bound to SOC n, is confirmed by the following: (i) small absolute Dd/DT values of the DNH and QNH amide protons; (ii) magnetically nonequivalent protons of the R side chain, strong downfield shift of the RN eH signal, large chemical shift difference of the magnetically nonequivalent DC bH 2 protons; and (iii) fairly comparable NOE pattern (intense NOE connectivities between successive protons YNH/DNH and DNH/ QNH). The occurrence of an ionic interaction between guanidinium and D-b-carboxylate groups and the formation of a type I b-turn involving the QNH3 RCO interaction, assumed in all SRYD containing sequences, are maintained after ligation of the gp63– SRYD peptides to SOC n (18, 27). In Table 3 are given the chemical shifts of the Arg and Asp side chains of the SRYD segment in the free and bound state. 2. Prevalence of One Antigenic Peptide’s Conformer after Anchorage to SOC n

FIG. 6. Reactivity of sera with various autoantibody specificities and nomal human sera in the anti-(PPGMRPP) 5–SOC 5 ELISA. Reprinted from J. Immunol. Methods, in press, C. Petrovas, P. G. Vlachoyiannopoulos, A. G. Tzioufas, C. Alexopoulos, V. Tsikaris, M. Sakarellos-Daitsiotis, C. Sakarellos, and H. M. Moutsopoulos, Copyright Elsevier, with permission from Elsevier Science.

Conformational analysis of the PPGMRPP epitope of the Sm and U1RNP antigens shows the presence of at least three conformers. The main conformer A (62%) is stabilized by an ionic interaction between the guanidinium and the C-terminal carboxylate groups, the carboxy-terminal peptide bond adopts the cis form, and the N-terminal sequence (PPGM) assumes a b II turn. Conformer B (21%) is also stabilized by an ionic interaction similar to that for conformer A, while the N-terminal part does not display any folded structure. Conformer C (17%) attains a completely extended structure. The multiple conformers of PPGMRPP may offer some explanation for the reactivity of sera with autoantibodies other (anti-Ro/La) than anti-Sm and anti-U1RNP (24). Coupling of the PPGMRPP peptide to SOC 5, through conversion of the C-terminal carboxylate group into the amide form, affects significantly the conformation of the antigenic peptide. Thus the bound form of the PPGMRPP peptide adopts a completely extended conformation (.95%), similar to the minor conformer of the free form of PPGMRPP and the main conformer of PPGMRPP-NH 2. The prevailing extended structural pattern is attributed to the absence of ionic interactions between the Arg-guanidinium and carboxylate group (28). The NH region of PPGMRPP (I), PPGMRPP-NH 2 (II), and (PPGMRPP) 5–SOC 5 is illustrated in Fig. 4.

ANTIGENIC/IMMUNOGENIC PEPTIDE CARRIERS

Biological Studies Using SOC n Conjugates as Antigens or Immunogens 1. [Ala 76]MIR–SOC n (n 5 2–7) Soluble MIR–SOC n are found to be at least as efficient as the MIR monomer in inhibiting, after preincubation, two anti-MIR mAbs from binding to the immobilized MIR peptide, thus suggesting a specific antibody recognition. It was also found that the SOC n carrier bearing four or five copies of the antigenic determinant experiences up to a 10-fold increase in MIR binding capacity (Fig. 5). These findings indicate a clear advantage in using MIR–SOC n, rather than MIR peptides, as potent antigens in diagnostic assays or in elaborating specific resins for antibody depletion (18, 19). 2. gp63–SRYD–SOC 6 Antisera obtained from rabbits immunized with gp63–SRYD–SOC n (n 5 5,6) are tested for their specific reactivity in ELISA against gp63–SRYD peptide and purified gp63. The two SOC n conjugates give satisfactory antibody titers versus the gp63–SRYD octapeptide, and they turn out to be better immunogens when compared with the same peptides conjugated to rabbit serum albumin. An increase of about 2-fold to 4-fold is observed in their titers. One also may note that antigp63–SRYD–SOC n (n 5 5,6) antibodies recognized purified Leishmania gp63 and the gp63 protein on intact parasites. It is concluded that the gp63–SRYD–SOC n conjugates are effective immunogens, recognizing their cognate protein, and they could possibly induce protective immunity against leishmaniasis. Immunizations with the SOC n carrier alone show that it is not immunogenic (18, 27). 3. (PPGMRPP) 5 –SOC 5 as Antigen An ELISA has been developed using as antigen the PPGMRPP peptide, anchored in five copies to SOC n, for

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evaluating the specificity and sensitivity of the assay to detect anti-Sm antibodies. Sera with different autoantibody specificities as anti-Sm, anti-U1RNP, antiRo(SSA)/La(SSB), ANA 1/ENA 2 (antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens) and normal human sera were examined (Fig. 6). RNA immunoprecipitation for the detection of anti-Sm and anti-U1RNP antibodies and counterimmunoelectrophoresis (CIE) for the detection of anti-Ro/ SSA and anti-La/SSB antibodies are used as reference techniques. The sensitivity of the ELISA is 98% and the specificity 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values are 82 and 86%, respectively. In ELISA experiments, using Sm/U1RNP purified complex as immobilized antigen, the sensitivity in detecting anti-snRNPs is 74%. It is concluded that (PPGMRPP) 5–SOC 5 is a better alternative compared with Sm and/or U1RNP, and that the reported ELISA is a convenient reproducible and sensitive screening test for anti-Sm/U1RNP reactivities (28, 29). 4. (PPGMRPP) 5 –SOC 5 as Immunogen With the aim of evaluating the immune response against the native form of Sm and U1RNP, rabbits are immunized with (PPGMRPP) 5–SOC 5, and high titers of antibodies to this conjugate can be detected in ELISA. However, antibodies recognizing the Sm antigen or precipitating the native structures of snRNPs do not appear in the sera of the immunized animals. These animals develop reactivity against a 67-kDa protein, which does not correspond either to the Sm or to the 70-kDa U1RNP antigen. We conclude that immunizations with (PPGMRPP) 5–SOC 5 result in a rather site-specific response than expansion of the B-cell repertoire. It is of interest that the immune response induced by the PPGMRPP conjugate is associated with immune-mediated kidney injury, suggesting that these

FIG. 7. Reactivity of sera from immunized rabbits with (La/SSB 289 –308) 4–SOC 4 (epitope 2) against immobilized rLa/SSB in ELISA.

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antibodies are crucial in inducing renal disease. The immune response against this epitope remains high even 9 months after the final boost, until the animals are sacrificed (30). 5. (La/SSB Epitope) 4-SOC 4 as Antigen The most sensitive and specific epitope of La/SSB, GSGKGKVQFQGKKTKF (349 –364), is coupled in four copies to the SOC 4 carrier and used for immunoassay development. Assays based on the recombinant La/ SSB are compared with (La/SSB 349 –364) 4–SOC 4. Of antiLa/SSB positive sera, 88% are reactive with both the SOC 4 conjugate and the recombinant La/SSB. Using sera anti-Ro/SSA positive but anti-La/SSB negative, the specificity of the (La/SSB 349 –364) 4–SOC 4 is found equal to 90%, while that of the recombinant La/SSB is only 63%. These results suggest that the La/SSB epitope (349 –364) anchored to SOC 4 exhibits high sensitivity and specificity and can be used as a reliable antigenic substrate in ELISA and dot blot tests (26). 6. (La/SSB Epitope) 4–SOC 4 as Immunogen Evaluation of the anti-La/SSB immune response is performed in immunized animals with (La/SSB epitope) 4–SOC 4. High titers of antipeptide antibodies are raised in the sera of all the animals. These antibodies can bind strongly each particular peptide immunogen. Furthermore, extension of the antigenic recognition to the other epitopes of the same protein occurs, as confirmed by inhibition experiments. Of interest is the induction of immune response to the whole La/SSB after immunization with each of the (La/SSB epitope) 4–SOC 4. These findings suggest an antibody spreading to all the epitopes, as well as to the intact antigen (31). For instance, sera obtained from rabbits immunized with (La/SSB 289 –308) 4–SOC 4 (Epitope 2) bind rLa/SSB strongly in ELISA (Fig. 7). The immune response against the immunizing epitopes remains high even 12 months after the final boost until the animals are sacrificed. Which is the role of SOC n in the augmentation of the immune response against the attached B-cell epitopes? Two possibilities are currently under investigation: i. The (B-cell epitope) n–SOC n construct is composed of several copies of a B-cell epitope (or autoepitope) anchored to a backbone composed of SOC n, therefore it resembles a T-cell-independent type 2 antigen (32); thus, the immunization process does not follow the hapten– carrier conjugate paradigm (33). Rather, it seems that multiple crosslinking of the B-cell receptor by such thymus-independent-type 2-like antigen can lead to autoantibody production either in the absence of or augmented by T-cell help. It is important to note that T-cell activation in this model takes place by a still unknown T-cell-receptor-independent process. ii. The T-cell receptor repertoire is as extended as

the B-cell receptor repertoire. Therefore, there is a chance that at least the linear B-cell epitopes of some antigens (or autoantigens) can be recognized by specific T-cell receptors. In other words, some linear B-cell epitopes of antigens (or autoantigens) may also function as T-cell epitopes. In that case the construct (B-cell epitope) n–SOC n provides a “molecule” of critical size and structure that can be taken, processed, and presented by B cells.

CONCLUDING REMARKS The major goal in the design of the presented oligopeptide carriers (SOC n) was to construct an artificial support with structural rigidity and regularity, so that peptide epitopes could be anchored without conformational restrictions and steric hindrance. 1H NMR studies and molecular modeling show that SOC n adopts a distorted 3 10-helical structure, which allows a favorable orientation of the lysine side chains and therefore of the attached peptides. Conformational analysis by 1H NMR spectroscopy of the SOC n conjugates points out that the peptides anchored to SOC n retain their original “active” conformation, or that the carrier imposes the prevalence of one conformer, thus confirming our initial design. The SOC n conjugates, when used as antigens, display significant biological reactivity, and the immunoassays developed were sensitive, convenient, and reproducible for screening antibody specificities related to autoimmune diseases. It is very probable that the helicoid structure of SOC n offers an optimal epitope presentation and helps the reconstruction and/or mimicking of the native epitopes. Immunizations with the SOC n conjugates generate in animals high titers of antibodies recognizing in all cases the immunogen peptide. Depending on the peptide anchored to SOC n, it can be identified either as an immune spreading covering various peptide sequences on the protein, as well as the intact protein, or a limited expansion of the B-cell repertoire. Multiple crosslinking of the B-cell receptor either by the B-cell epitopes coupled to SOC n or uptake by B cells and presentation to T-cell receptors that cross-react with these B-cell epitopes are two alternative explanations for the mode of action of (epitope n)–SOC n conjugates and the continuous secretion of specific antibodies long after immunization.

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