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Biologicals (2001) 29, 197–207 doi:10.1006/biol.2001.0305, available online at http://www.idealibrary.com on Manipulation of Epitope Function by Modification of Peptide Structure: A Minireview Ferenc Hudecz Research Group of Peptide Chemistry, Hungarian Academy of Science, Eötvös L. University, P.O. Box 32, Budapest 112, Hungary, H-1518 Abstract. We have explored various approaches to modify the immunrecognition of linear peptides representing sequential or continuous topographic B-cell or T-cell epitopes. For these studies, epitopes from herpes simplex virus (HSV) glycoprotein D (gD) and from mucin 1 and mucin 2 glycoproteins or T-cell epitopes from 16 kDa and 38 kDa proteins of Mycobacterium tuberculosis were selected. To increase antigenicity and immunogenicity we have prepared cyclic and chimaeric peptide variants as well as epitope peptides with altered flanking regions and epitope-carrier conjugates containing multiple epitope © 2001 The International Association for Biologicals copies. Key words: epitope peptide-conjugates, epitope-chimaera, epitope flanking, synthetic peptide antigens, mucin antibody epitopes, protection against HSV infection, epitopes of M. tuberculosis proteins. Introduction Linear peptides representing sequential or continuous topographic B-cell or T-cell epitopes could be poorly recognised by antibodies or T-cells specific for the protein. To increase immunoreactivity, including antigenicity and immunogenicity of peptides belonging to the above classes of B-cell or T-cell epitopes, several experimental approaches have been investigated. Since B-cell epitope sequences are frequently localised in -turns or loop regions of a protein, the corresponding cyclic peptide could be a logical and better mimicry of the native secondary structure than the linear oligopeptide. Similarly, the stabilisation of steric, mainly secondary structure of a B-cell epitope could be achieved by insertion of the sequence into an appropriate site of a ‘‘host’’ protein sca#old. Thus the ‘‘guest’’ B-cell epitope in chimaeric peptide could be more e#iciently recognised by epitope-specific antibodies compared with the linear oligopeptide. Specific T-cell responses induced by peptides containing a minimal size functional T-cell epitope could be modulated by the Address all correspondence to: Professor Ferenc Hudecz, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, Budapest, Pázmány P. sétány. 2., H-1117, Hungary. Fax: (36)-1-372-2620. E-mail: [email protected] 1045–1056/01/090197+11 $35.00/0 appropriate replacement of amino acid residues in the epitope core and/or by the alteration of the flanking regions connected to the N- and/or C-terminal of the core. This approach could lead to the development of T-cell antagonist/agonist compounds. Another strategy for increasing the sensitivity of antigen binding or the immunogenic properties is the multiplication of copies of the same or defined number of di#erent B- or T-cell epitopes of microbial or tumour origin. To achieve this polymerised epitope peptides could be prepared. Alternatively, covalent epitope-carrier conjugates could be synthesised using optimal size oligopeptides representing functional epitopes and protein or synthetic carriers (e.g. KLH, BSA, branched chain polymeric polypeptides, multiple antigenic peptides (MAP), a sequential oligopepide carrier (SOC), oligotuftsin). These approaches not only provide a better understanding of the antigenic structure of proteins, but also contribute to the development of synthetic antigens as artificial vaccines or diagnostic reagents. In this communication a brief overview will describe our recent results with (i) epitopes from herpes simplex virus (HSV) glycoprotein D (gD); (ii) epitopes from mucin 1 and mucin 2 glycoproteins and (iii) with T-cell epitopes from 16 kDa and 38 kDa proteins of Mycobacterium tuberculosis. 2001 The International Association for Biologicals 198 F. Hudecz SALLEDPVG-NH2 CSALLEDPVG-NH2 I CSALLEDPVG-NH2 II H-YCCNPVACGRHYSC-NH2 III H-SALLEDPVGK-NH2 H-CSALLEDPVGK-NH2 IV V 1 α-[Tyr ]-conotoxin H-YCCNPVACGDPVGC-NH2 1 bicyclic HSV-α-[Tyr ]-conotoxin H-YCCNPVACGPDTRC-NH2 1 bicyclic MUC1-α-[Tyr ]-conotoxin Figure 1. Schematic representation of cyclopeptides containing the 281DPVG284 epitope sequence from glycoprotein D of herpes simplex virus in endo (IV, V) or in exo (I–III) position. Figure 2. Primary structure of -[Tyr1]-conotoxin and its chimaera derivatives containing epitope sequences from glycoprotein D of herpes simplex virus (281DPVG284) or from mucin 1 glycoprotein (PDTR). Cyclic and chimaeric peptides IgG type antibody responses showed that the bi-cyclic HSV--[Tyr1]-conotoxin chimaera induced strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice (Fig. 3). Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognise the DPVG motif presented in the bi-cyclic HSV-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimaera. We also found that the IgM monoclonal antibodies are able to recognise the linear DPVG sequence, while the majority of the IgG antibodies are directed to the same motif in a conformation stabilised by double cyclisation. It is interesting to note that the bi-cyclic HSV--[Tyr1]conotoxin chimaeric peptide and native -conotoxin GI showed similar CD spectra in PBS, which might suggest that these compounds also share similar secondary structures. Using the same -conotoxin GI sca#old we have also synthesised a chimaeric peptide with an inserted Pro-Asp-Thr-Arg (PDTR) epitope of the mucin 1 glycoprotein (MUC1) instead of the native 8 Arg-His-Tyr-Ser12 tetramer (Fig. 2). MUC1 is expressed and often secreted by epithelial cells and contains a polypeptide core consisting of a variable number (30–100) of repeats of a 20 amino acid sequence, APDTRPAPGSTAPPAHGVTS. Carcinoma cells produce MUC1 that is underglycosylated when compared to the version expressed by normal resting cells. The incomplete glycosylation exposes a normally cryptic polypeptide core whose amino acid sequences could be recognised by the immune system as epitopes. The appearance of these epitope domains is largely restricted to cancer cells; therefore MUC1 is an attractive target for cancer immunodiagnosis and immunotherapy. It has been demonstrated earlier that the PDTR sequence comprises the minimal epitope for MUC1 specific monoclonal antibodies HMFG1 (PDTR) and HMFG2 (DTR). Hydropathicity and secondary structure HSV, with its two closely related serotypes (HSV-1 and HSV-2), is one of the most common infectious agents in humans. Glycoprotein D represents a major immunogenic component of the virion envelope. Using prediction analysis of the sequence of gD from HSV-1 and synthetic peptide-conjugates with branched polypeptide poly[Lys(DL-Alam)], (AK) where m3) the 281DPVG284 tetramer sequence has been identified as the core of the epitope within the highly hydrophilic 276–284 region possessing a -turn. Several cyclic versions of S276ALLEDPVG284 nonapeptide were prepared (Fig. 1) consisting of either (a) a head-to-side-chain lactam ring, where the DPVG motif is situated essentially outside the ring (exo-form) (I–III) or (b) a side-chain-to-side-chain lactam ring between the -carboxyl group of Glu and the -amino group of a Lys residue attached to the C-terminus of the sequence. In the latter case the DPVG motif is a part of the six-residue lactam ring (endo-form) (IV–V). The CD studies together with NMR data have indicated that the conformation of the peptides is highly dependent on the relative position of the DPVG epitope in the cyclic HSV peptides.1,2 Antibody binding studies are in progress and the results will be published elsewhere. The DPVG core epitope has been inserted as ‘‘guest’’ sequence in the ‘‘host’’ structure of -conotoxin GI, a 13 residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The -conotoxin GI was selected as a presenting sca#old since it also contains a -turn in the 8–12 region which is stabilised by two disulphide bridges in positions 2–7 and 3–13. Thus the tetramer sequence of -conotoxin, 8Arg-His-TyrSer12 has been replaced by DPVG from the gD of HSV (Fig. 2). The linear, the monocyclic and the bi-cyclic forms of the chimaera have been synthesised and their immunogenicity was compared.3 The characteristics of the primary and memory IgM and Immunoreactivity of modified peptide epitopes 100 1200 (A) (A) CBA BALB/c C57B1/6 1000 80 800 60 Inhibition % OD492 199 600 400 40 20 200 0 0 102 103 104 105 lg serum dilution 106 107 –20 0.0001 1200 (B) 60 Inhibition % OD492 10 80 600 400 200 102 0.001 0.01 0.1 1 Peptide concentration (mmol/l) (B) 800 0 10 100 CBA BALB/c C57B1/6 1000 0.001 0.01 0.1 1 Peptide concentration (mg/ml) 40 20 103 104 105 lg serum dilution 106 107 Figure 3. Antibody response induced by bi-cyclic HSV-Tyr1]-conotoxin chimaera in C57/Bl/6, CBA and BALB/c mice. Binding of polyclonal antibodies to the bi-cyclic HSV--[Tyr1]-conotoxin chimaera (A) and to [DPVG]-AK conjugate (B) target. prediction analysis confirmed by CD and NMR experiments and supported by independent computational non-restrained study have identified a type I -turn in the PDTR region. Based on these considerations we have prepared all three forms of the chimaera (linear, monocyclic, bicylic) and compared their antibody binding properties.4 As documented by the CD spectra, the bi-cyclic MUC1--[Tyr1]conotoxin chimaera peptide showed a partially ordered conformation with a -turn character. In antibody binding studies the RIA data showed that the MUC1--[Tyr1]-conotoxin chimaera was recognised by monoclonal antibody HMFG1 specific for the PDTR sequence, while no binding was observed between monoclonal antibody HMFG2 and 0 –20 0.0001 Figure 4. Inhibition of the binding of monoclonal antibodies to [CAPDTRPAPG]-AK target antigen (A) or of HMFG1 monoclonal antibody to [KAPDTRPAPG]-BSA (B) with MUC1--[Tyr1]-conotoxin chimaera peptides in competition RIA. Monoclonal antibody HMFG2 with linear peptide MCnTx1 (), or with bi-cyclic peptide (). Monoclonal antibody HMFG1 with linear peptide MCnTx1 (), monocyclic (), or with bi-cyclic peptide () and control peptide APDTRPAPG (). various forms of the chimaera (Fig. 4A). Significant di#erences were found in the HMFG1 recognition of the PDTR epitope among the three forms of the chimaera (Fig. 4B). HMFG1 using two di#erent target antigens (synthetic epitope-conjugate or native MUC1) recognises the PDTR more e#iciently in the linear than in the bi-cyclic compound, but no reactivity was found with the monocyclic forms 200 F. Hudecz Table 1. Amide I peaks in the FT-IR spectrum and monoclonal antibody 996 binding of MUC2 peptide Peptide 16 PTPTGTQ22 pTPTGTQ22 16 ptPTGTQ22 16 ptpTGTQ22 16 ptptGTQ22 16 High frequency region cm 1 (%) Solvated amides cm 1 (%) -turns cm 1 (%) -turns cm 1 (%) 1631 (4) 1630 (1) 1661 (5) 1660 (11) 1646 (15) 1644 (14) 1643 (17) 1643 (8) 1641 (8) 1673 (43) 1674 (46) 1674 (46) 1675 (37) 1675 (36) 1624 (3) 1625 (3) IC50* (mol/l) 3·8 6·5 33 160 6400 *IC50 is the peptide concentration required for 50% inhibition of 996 antibody binding to BSA-[K12VTPTPTPTGTQTPT25] target antigen. of MUC1--[Tyr1]-conotoxin chimaera, underlining the importance of certain conformers stabilised by double cyclisation. Examples outlined above might indicate that both the antibody binding and immunogenic properties of the linear B-cell epitope could be improved by restriction of the number of conformers present in their linear form. However it is also clear that there is a need for an appropriate design of the cyclic or chimaeric version of the epitope. Small or even minute changes in the position of the epitope in the ‘‘presenting’’ sca#old could lead to a dramatic alteration of antibody binding and/or immunogenicity. Replacement of amino acid residues in the epitope flanking regions Effect of D-amino acid substitution in a mucin 2 glycoproteion (MUC2) epitope on MUC2 specific monoclonal antibody recognition. The majority of published work reports on the replacement of all the -amino acid residues in a peptide by the -enantiomers leading to normal (in all--isomer peptides) or reversed (in retro-all--isomer peptides) amide linkage. Relatively little has been communicated on the antibody recognition of peptides partially substituted by -amino acid residue(s) and to the best of our knowledge no data are available on the antibody binding to epitope peptides containing -amino acid residues in the flanking region. In model experiments we aimed to perform a systematic analysis to what extent could we substitute the N-terminal flanking region of the epitope by -amino acids without significant decrease in antibody recognition. For this we have selected a B-cell epitope from MUC2 whose applicability as a tumour marker in colon carcinoma patients is under investigation. Our previous studies showed that the core of the epitope recognised by the protein specific monoclonal antibody (MAb 996) within the PTTTP ITTTTTVTPTPTPTGTQT tandem repeat unit of MUC2 glycoprotein is the 19TGTQ22 sequence. However, for optimal binding the 16PTPTGTQ22 sequence is required.5 We have studied the influence of -amino acid substitution in the flanking region on the antibody recognition of the 19TGTQ22 epitope core.6 Analogue peptides corresponding to the optimal epitope sequence (16PTPTGTQ22) have been prepared by the replacement of single or multiple -amino acid residues at the N-terminal part of the molecule (Table 1). According to previous studies this portion of the all- 16PTPTGTQ22 peptide possesses a -turn secondary structure important for e#icient monoclonal antibody interaction.7 The binding properties of sequentially modified peptides (pTPTGTQ, ptPTGTQ, ptpTGTQ and ptptGTQ) have been analysed by a MUC2 glycoprotein specific monoclonal antibody (MAb 996) using an RIA inhibition assay and characterised by IC50 values. At the same time we have investigated the secondary structure of the compounds by CD and Fourier-transform infrared spectroscopy in solution. Our data showed that the presence of amino acid residue(s) at position(s) 16P, 16PT17 or 16 PTP18 resulted in gradually decreasing antibody binding, but the replacement of the -Thr at position 19 almost abolished the activity (Table 1). Parallel with this reduction, changes in the conformer population have been detected. The propensity of the pTPTGTQ peptide to adopt a folded, most probably -turn, structure in water can be correlated with its essentially preserved antibody recognition. After Immunoreactivity of modified peptide epitopes Effect of non-native flanking regions in Mycobacterium tuberculosis epitope peptides on 38 kDa glycoproteion specific T-cell recognition. T-cells play a critical role in the development of protective and pathogenic immune responses against M. tuberculosis infection. Identification of immunodominant epitopes could provide a rational basis for the construction of synthetic peptide-based diagnostic reagents. It is known that the T-cell stimulatory activity of naturally processed proteins or synthetic peptides depends not only on the sequence of the T-cell epitope core, but also on its flanking residues. The 38 kDa glycosylated lipoprotein, a secreted constituent of M. tuberculosis, is immunogenic in active tuberculosis. A peptide representing the 65–83 (FNLWGPAFHERYPNVTITA) region was found to be one of the immunodominant T-cell stimulatory domains in humans and in 57CBL/10 mice pre-sensitised with either live or killed organisms of the M. tuberculosis complex or with recombinant 38 kDa. The murine CD4 + T cell epitope core of this region was localised to amino acid residues 75–81 (RYPNVTI) by deduction from PEPSCAN analysis using overlapping 15-mer peptides. To clarify the role of flanking regions adjacent to the epitope core a peptide representing the deduced core was prepared with extensions at both N- and C-termini.8 These flanks are composed of either amino acid residues from the native sequence and terminated by Ala and/or Ser residues or oligopeptides of Ala or Ser exclusively. Their binding to isolated H-2-Ab MHC glycoprotein as well as their T-cell stimulatory capacity were assayed using a specific murine hybridoma T cell line [38.H6], lymph node cells from the native 20-mer peptide primed 57 CBL/10 mice, and human PBMCs from sensitised individuals.9 The stimulation of the 65–83 specific T-cell hybridoma was induced by the full-length FNLWG PAFHERYPNVTITA peptide, but not by the 1200 1000 800 S.I. further substitution the peptide still contained and/or -turn folded secondary structural elements, but in a significantly smaller conformer population and built up from -residues. These data suggest that for significant MAb 996 recognition, the N-terminal flanking region next to the core epitope could contain at least one -amino acid without significant loss of binding activity and folded conformation required. These findings might be useful for the design of artificial epitopes, peptide-vaccines with increased enzymatic stability and extended biological half-life. 201 600 400 65–83 AASA(75–81)AAAA AAAA(75–81)AAAA AAA(74–82)AAA 74–81 200 0 0.1 1 10 100 c (µM) Figure 5. The e#ect of non-native flanking regions on the T-cell recognition of peptides containing the deduced core. 65FNLWGPAFHERYPNVTITA83 specific hybridoma cells [38.H6] were incubated with peptides at 0·210 6 to 5010 6 in the presence of irradiated 57 CBL/10 spleen cells. Culture supernatants were tested with the cell line HT2. The results are expressed as stimulation indices (SI=cpm with peptide/cpm without peptide). The average background value was 555·4 cpm. eight-mer peptide 74ERYPNVTI81, being only one amino acid longer at the N-terminus than the deduced murine T-cell epitope core (Fig. 5). Elongation of the deduced core by four Ala residues at both N- and C-termini in AAAARYPNVTIAAAA (A4-7581-A4) induced significant stimulation throughout the concentration range studied. Peptide AAAERYPNVTITAAA (A3-74-82-A3), having an extended core from the native protein, stimulated somewhat less than that of peptide 65–83 (Fig. 5). Interestingly a substitution of one Ala to Ser in the N-terminal flank increased the T cell stimulatory capability significantly above that stimulated by the native 20-mer (65–83). Peptide AASARYPNVTIAAAA (A2SA-75-81-A4) containing only the deduced core was more potent than its counterpart (Fig. 5). It should be noted that this construct was even more active than peptide 65–83, which contains the whole immunodominant domain. To test the in vivo immunogenicity of peptides 65–83 and A2SA-75-81-A4, 57CBL/10 mice were immunised and LN cells were then stimulated in vitro by the homologous and heterologous peptides. Although both peptides were immunogenic in vivo, immunisation with peptide A2SA-75-81-A4 resulted in stronger immune responses when LN cells were challenged in vitro with either the native 65–83 202 F. Hudecz capacity of the deduced, and non-functional epitope core. 100 Conjugation to branched chain polypeptide Inhibition % 80 60 40 CLIP AAAA(75–81)AAAA AASA(75–81)AAAA AA(73–83)AA 65–83 20 0 0.1 1 10 100 c (µM) Figure 6. The e#ect of non-native flanking regions on the binding to the H-2-Ab molecule. Inhibition of binding of the biotinylated CLIP peptide (0·7 ) to H-2-Ab by 65 FNLWGPAFHERYPNVTITA83 and its analogues. Inhibition with the unlabelled CLIP peptide is shown as a reference. The values represent the means of triplicate wells from a single experiment, which was repeated three times. Variations were <20% of the mean. peptide or its non-native flanked analogue, A2SA-7581-A4. These results demonstrate that peptide A2SA75-81-A4 is not only more e#iciently recognised by T cells in vitro, but it also has improved in vivo immungenicity compared with the native 20-mer peptide 65FNLWGPAFHERYPNVTITA83. The peptides were tested for binding to isolated H-2-Ab glycoproteins using VSKMRMATPLLM QALP (CLIP) as a reference peptide. As shown in Figure 6, peptide 65–83 binds to H-2-Ab with somewhat lower a#inity than CLIP. Replacement of native flank region with alanine as in the A4-7581-A4 peptide resulted in a relatively small decrease in binding. The peptide with Ser introduced into the N-terminal flank (A2SA-75-81-A4) showed binding similar to that of the native peptide (IC50 =0·9 ). Therefore, when normalised for the experimental conditions, the a#inity (Ki) of the native 65–83 peptide is 0·158 , whereas a#inities of peptides with artificial flanks are in the range 0·07–0·29 . Taken together, this is the first study in the literature in which a synthetic peptide constructed by elongation of a non-functioning deduced epitope core with short, simple flanking segments proved to be a stronger immunogen than the peptide which contains the natural adjacent amino acid residues. These findings suggest that non-native flanking regions are able to enhance the T-cell stimulatory New groups of branched chain polymeric polypeptides were developed in our laboratory with the general formula poly[Lys(Xi--Alam)] (XAK), poly[Lys(Xi--Serm) (XSK), or poly[Lys(-Alam-Xi)] (AXK), where i<1, m3, and X represent an additional optically active amino acid residue.10–12 These compounds are poly[-Lys] derivatives substituted by short (three to six amino acid residues) branches at the -amino groups. The side-chains are composed of about three -Ala (Fig. 7) or -Ser residues and one other amino acid residue (X) at the N-terminus of the branches (XAK or XSK) or next to the poly[-Lys] backbone (AXK). These compounds were characterised by their size, chemical structure (primary structure, solution conformation) and their biological properties (in vitro cytotoxicity, pyrogenicity, biodegradation, immunoreactivity and biodistribution). Under physiological conditions (pH 7·3 in 0·15 NaCl) depending on the identity of amino acid X, branched polypeptides could possess polycationic (e.g. poly[Lys(Leui-Alam)], (LAK) or poly[Lys(Orni--Alam)], (OAK)), amphoteric (e.g. poly[Lys(Glui--Alam)], (EAK) or polyanionic (e.g. poly[Lys(Ac-Glui--Alam)], (Ac-EAK) or poly[Lys(Suc-Glui--Alam)]) (SucEAK) character. We have demonstrated that the composition of the side chains and the charge properties of these polypeptides determine their solution conformation, phospholipid membrane interaction and various biological e#ects (e.g. cytotoxicity, blood clearance, tissue distribution, immunreactivity). These macromolecules were used for the synthesis of B-cell epitope peptide conjugates to be used as target antigens for the specific and sensitive detection of MUC1 glycoprotein-specific antibodies.13 More recently this class of polymeric polypeptides was conjugated with several epitope peptides derived from glycoprotein D of HSV or from M. tuberculosis. We have shown that the composition of the polymeric component has a marked influence on the immuno-recognition of covalently attached epitope peptide, and also on the interaction between phospholipid mono- or bilayers and epitope conjugates.14 Carrier-dependent induction of HSV gD epitopespecific, protective immune response. To investigate the roleof a macromolecular carrier in inducing an Immunoreactivity of modified peptide epitopes + NH3 + NH3 - CH3 Leu CH2-CH-(CH3)2 - CH3 Ser/Thr + NH3 203 - CH3 Orn/Lys - CH3 + NH3 + NH3 OH + NH3 + - CH3 - CH3 - CH3 poly[Lys(Leui-DL-Alam)] LAK - CH3 NH3 - CH3 poly[Lys(Xi-DL-Alam)] SAK (X = Ser), TAK (X = Thr) poly[Lys(Xi-DL-Alam)] KAK (X = Lys), OAK (X = Orn) + NH3 + NH3 - CH3 oligo(DL-Ala) B X - CH3 - CH3 poly[L-Lys] – + + NH3 CO-(CH2)2-COO poly[Lys(Xi-DL-Alam)] XAK Glu + NH3 - CH3 NH3 – COO - CH3 - CH3 - CH3 - CH3 + NH3 NH-CO-CH3 – COO Suc-Glu - CH3 – - CH3 poly[Lys(Glui-DL-Alam)] EAK NH COO - CH3 Ac-Glu - CH3 poly[Lys(SucGlui-DL-Alam)] Suc-EAK poly[Lys(AcGlui-DL-Alam)] Ac-EAK Figure 7. Schematic representation of the branched chain polymeric polypeptides. epitope-specific, protective immune response against viral infection, artificial antigens with branched polypeptide carrier and B-cell epitopes have been designed (Fig. 8). Peptides corresponding to two di#erent epitope regions (KYALADASLKMADPNRFRG KDLP, 1–23 and SALLEDPVG-NH2, 276–284) of glycoprotein D of HSV type 1 were conjugated with two representatives of branched polypeptides (AK and LAK) and KLH.13 Under the conditions used for coupling the sidechains of Asp and Glu could be involved in amide bond formation, but only one carboxyl group of the gD-1 peptides was linked to the -amino group of the terminal alanine (AK) or leucine (LAK) residue. 204 F. Hudecz oligo(DL-Ala) poly[L-Lys] 276 SALLE...284 1 K....P23 276 Figure 8. Schematic representation of the branched polypeptide conjugate of SALLEDPVG284-NH2]-AK and KYALADASLKMADPNRFRGKDLP23]-AK containing epitope sequence from glycoprotein D of herpes simplex virus. 1 CBA or BALB/c mice were immunised with conjugates and relevant controls in CFA. The magnitude of the peptide-, conjugate- and carrier-specific Ab responses were then analysed and the survival of animals infected with a lethal dose of 50 HSV-1 was investigated. AK conjugates ([1–23]-AK and [276–284]-AK) induced significant and comparable gD-epitopespecific IgG responses accompanied by the appearance of a low level of carrier-specific antibodies. In contrast, negligible epitope-specific IgG responses were elicited with [1–23]-LAK or [276–284]-LAK. Immunisation with the respective peptide-KLH conjugates induced intense carrier-specific response without measurable peptide specificity. In a protection experiment (Fig. 9) carried out in BALB/c mice, repeated administration of LAKbased conjugates ([276–284]-LAK or [1–23]-LAK) were not able to prolong survival significantly compared to the free peptide or untreated control mice. In sharp contrast, pre-immunisation with the [1–23]AK or [276–284]-AK conjugate resulted in complete protection of a considerable proportion (50%) of animals against a 100-fold lethal dose of HSV-1. These results demonstrated that it is feasible to construct synthetic immunogens with synthetic branched polypeptide carriers which possess higher e#icacy to induce epitope-specific antibody responses than KLH-based conjugates. Comparative studies with branched polypeptide carriers and two epitope peptides from gD suggest that the capability of eliciting protective immune response against HSV-1 infection is dependent on the carrier molecule. Enhancement of the T-cell response to a mycobacterial peptide by conjugation to a synthetic branched polypeptide. T-cell epitopes containing peptides covalently attached to macromolecular carriers can be considered as synthetic immunogens for the development of skin-test diagnostics and of vaccines. To investigate the role of the carrier on the recognition of an attached T-cell epitope, we have prepared conjugates containing peptide 350DQVHFQPLPP AVVKLSDALI369 representing a T-cell epitope domain of 38 kDa protein of M. tuberculosis and branched chain polypeptides EAK, Ac-EAK or SucEAK (Fig. 7). In vitro T-cell immunogenicity of these three conjugates together with relevant free peptide, free branched polypeptide as well as their mixture was studied using human peripheral blood mononuclear cell (PBMC) cultures from healthy subjects and from tuberculosis patients.15 We found that interferon gamma production as well as T-cell proliferation increase was dependent on the carrier compound. A conjugate containing Suc-EAK enhanced IFN- production more than 13-fold (from 22·6 to 294 pg/ml, P=0·001) in PBMCs from healthy individuals, and 8·7-fold (P=0·012) in cells from tuberculosis patients. In a proliferation assay the stimulation index was elevated, when Ac-EAK or Suc-EAK was present in the conjugate (Fig. 10). These data clearly suggest that the selection of the carrier (Suc-EAK>Ac-EAK) greatly Immunoreactivity of modified peptide epitopes 205 20 (A) 100 15 60 [1–23]-AK 40 S.I. Survival % 80 1–23 20 control 2 0 10 [1–23]-LAK 4 6 8 10 12 14 Time after HSV-1 infection (days) 30 5 (B) 100 Survival % 80 0 0.1 60 [276–284]-AK 40 273–284 20 control 0 2 [276–284]-LAK 4 6 8 10 12 14 Time after HSV-1 infection (days) 30 Figure 9. Survival of HSV-1 infected BALB/c mice preimmunised with gD-1 epitope peptide-branched polypeptide conjugates showing the e#ect of the carrier on the protection induced by [1–23]-AK, [1–23]-LAK conjugates or [1–23] peptide (A) or [276–284]-AK, [276–284]-LAK conjugates or [273–284] peptide (B). Control mice were injected with PBS. influences the T-cell immune response. epitope peptide-specific Stimulation of T-cell responses with a conjugate containing two distinct epitopes attached covalently to a branched polypeptide carrier. Recently a fully syn- thetic prototype conjugate with two independent T-cell peptide epitopes of M. tuberculosis proteins (16 kDa and 38 kDa) was produced (Fig. 11). As a carrier, an amphoteric branched chain polypeptide, EAK with poly[-lysine] backbone, has been used. This polypeptide with free -amino and -carboxyl groups at the end of the side chains was conjugated with peptides representing two immunodominant 1 10 –6 Concentration × 10 100 Figure 10. E#ect of a polypeptide carrier on the reaction of human peripheral blood mononuclear cells to peptide 350 DQVHFQPLPPAVVKLSDALI369 from 38 kDa protein of Mycobacterium tuberculosis and its conjugates with Ac-EAK or Suc-EAK polyanionic branched polypeptide carrier (average of four experiments performed with four donors). The results are expressed as stimulation indices (SI=cpm with peptide/cpm without peptide). Positive response: SI>3. regions of the 16 kDa and 38 kDa proteins of M. tuberculosis, respectively.16 Peptide 91CSEFAYG SFVRTVSLPVGADE110 was elongated by Cys at the N-terminus and attached to the carrier containing protected SH groups to form disulphide bridges. Peptide 65FNLWGPAFHERYPNVTITA83 was conjugated to the 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP) modified and acetylated EAK by introducing an amide bond between the free -amino group of the peptide and the free -COOH group of Glu at the terminal position of the branches. This strategy lead to chemically well defined synthetic immunogens that contain two di#erent epitopes in multiple copies covalently linked to a synthetic branched polypeptide carrier. In this conjugate peptide 91 SEFAYGSFVRTVSLPVGADE110 from the 16 kDa protein and peptide 65FNLWGPAFHERYPNV TITA83 from the 38 kDa protein were conjugated. In vitro T-cell immunogenicity of a prototype conjugate together with relevant Ac-EAK-based 206 F. Hudecz NH- -S-S H2-NCO- CH3-CO- poly[L-Lys] 40 (A) 5 × 10–6 M 25 × 10–6 M Glu NH- oligo(DL-Ala) -CONH2 S.I. CH3-CO- ·S-S- H2NCO- CH3-CO- H2-Peptide1 20 150 AcEAK (93–110) (65–83) (B) 5 × 10–6 M 25 × 10–6 M 100 S.I. AcEAK (65–83) (91–110) 0 (91–110)-AcEAK 50 K2 conjugates was studied using T-cell hybridomas, lymph node cells from immunised mice and human PBMC cultures from PPD-positive individuals. Conjugate K2, the free peptides (91SEFAYG SFVRTVSLPVGADE110 and 65FNLWGPAFHERY PNVTITA83), their Ac-EAK conjugates and unsubstituted branched polypeptides (Ac-EAK and EAK) were tested using the murine T-cell hybridomas 38.I.6 and 16.10.7 (Fig. 12). The concentration of conjugates was calculated according to the respective peptide contents. The 65–83 specific T-cell hybridoma was recognised by conjugates K2 and 38.I-AcEAK as well as the free 65FNLWGPAFHERYPNVTITA83 peptide; as expected, peptide 91SEFAYGSFVRTVSLPVG ADE110 corresponding to the 16 kDa protein did not induce a response (Fig. 12A). The activity of peptide 91 SEFAYGSFVRTVSLPVGADE110 from the 16 kDa protein was investigated using T-cell hybridomas obtained from peptide 91–110 immunised mice. Peptide 91SEFAYGSFVRTVSLPVGADE110 conjugated either to EAK through a disulphide bond (in conjugate K2) or to Ac-EAK with an amide bond was able to induce T-cell stimulation (Fig. 10B). Specificity in both sets of experiments has been proved by the lack of e#ect on these cells of free 65–83 (Fig. 12B) or 91–110 (Fig. 10A) peptides or branched polypeptides (Ac-EAK or EAK). An alternative strategy for disulphide-based conjugation is the application of Cys(Npys) as a coupling agent containing Npys (3-nitro-2pyridinesulphenyl) group in the side-chain of cysteine. Cys(Npys) residues were used to modify branched polypeptides at the end of their branches and HS-peptide epitopes.17 0 (65–83)-AcEAK Figure 11. Schematic representation of conjugate K2 containing two di#erent epitope peptides from M. tuberculosis proteins 65FNLWGPAFHERYPNVTITA83 and 91CSEFAYGSFVRTVSLPV-GADE110 attached to branched polypeptide EAK by an amide or disulphide bond, respectively. K2 Cys-Peptide2 Figure 12. T-cell recognition of conjugate K2 by hybridoma [38.H6/1] specific for 65FNLWGPAFHERYPNVTI TA83 (A) and by hybridoma [16.10.7] specific for 91 SEFAYGSFVRTVSLPV-GADE110. Cells were incubated with conjugate K2 and control compounds at 510 6 and 2510 6 in the presence of irradiated 57CBL/10 spleen cells. Culture supernatants were tested with the cell line HT2. Results are expressed as stimulation indices (SI=cpm with peptide/cpm without peptide). These findings suggest that the conjugate K2 containing two distinct T-cell epitopes are able to induce epitope specific responses in hybridoma cells. Thus, the capability of epitope peptides to initiate specific T cell stimulation was preserved Immunoreactivity of modified peptide epitopes after their well-defined covalent attachment to a synthetic polymeric polypeptide. Acknowledgements Experimental work summarised in this paper was supported by grants from the from WHO (T9/181/133), Hungarian-Spanish Intergovernmental Programme (5/1998,3/2001), from the Hungarian Research Fund (OTKA No. T-3024, T-4217, T-014964, T-03838) and from the Hungarian Ministry of Education (FKFP No. 0101/97). 9. 10. 11. 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