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Journal of General Microbiology (1992), 138, 2197-2203. Printed in Great Britain 2197 Targeting of specific domains of diphtheria toxin by site-directed antibodies DOROTHEA SESARDIC,* VANITAKHANand MICHAEL J. CORBEL Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire EN6 3QG. UK (Received 19 March 1992; revised 15 June 1992; accepted 23 June 1992) ~ Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-l5 7 on fragment A, which contains the putative protein elongation factor (EF-2)ADP-ribosyltransferasesite; DTx224237 on fragment B, selected on the basis of forming a predicted surface loop; and D T x ~ on ~ fragment ~ - ~ B, ~ ~ forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region ia the native protein. The epitopes for two of the anti-peptideantibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominately against a narrow region within the peptide, consisting of only 5-6 aa residues. The predicted location of the peptide and their epitopes was confirmed by inspection of the X-ray crystallographic structure of DTx. Antibodies to peptides were selective for the toxin, one binding to DTx some 5-6O-foldbetter than to diphtheria toxoid, presumably reflecting variability caused by toxoid preparation at this epitope. None of the antisera produced protected against DTx challenge in the guinea pig intradermal test in aim. Although the availability of site-specific antibodies that recognize neutralizing epitopes would be very valuable, antibodies such as those described here should prove extremely useful in the structure-function analysis of DTx. Introduction Diphtheria toxin (DTx) is an extremely potent bacterial protein toxin with M , 58350, synthesized as a single polypeptide chain by pathogenic strains of Corynebacterium diphtheriae. Following mild trypsin treatment and reduction, DTx is cleaved into two functionally different fragments, A and B. Fragment A is a potent NAD+binding enzyme which ADP-ribosylates elongation factor 2 (EF-2) (Pappenheimer, 1977; Collier et al., 1964), thereby inhibiting protein synthesis. Fragment B, on the other hand, is required for recognition of specific cell surface receptors (Honjo et al., 1968; Uchida et al., * Author for correspondence. Tel. (0707) 54753; fax (0707) 46730. Abbreuiation : KLH, keyhole limpet haemocyanin. 1977), binding to which results in translocation of fragment A into the cytosol where it exerts toxicity (Cieplak et al., 1987; Mekada et al., 1988). The clinical manifestations of diphtheria are not due to invasive bacterial infection but to release of this potent cytotoxin. Thus, successful vaccination against diphtheria requires the acquisition of toxin neutralizing antibodies. There have been several suggestions as to how modern methods could be applied to create purer or more effective vaccines against toxin-producing bacteria, including those based on peptides capable of eliciting neutralizing antibodies (Audibert et al., 1981, 1982). The main obstacle in the development of such vaccines is lack of understanding of the immunological specificity of the areas of the toxin responsible for eliciting protective antibodies. Antibodies raised against peptides corresponding to the linear protein sequence 0001-7473 O 1992 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 2198 D . Sesardic, V . Khan and M . J . Corbel Table 1. The structure of the peptides chosen for synthesis and their corresponding regions on diphtheria toxin The amino acid sequence of diphtheria toxin was predicted from the cDNA sequence (Greenfield etal., 1983) and is numbered from the N-terminal glycine which is the first amino acid on fragment A. Peptide name DTA DTB 1 DTB2 Structure Sequence on DTx Ala-Glu-Gly-Ser-Ser-Ser-Val-Glu-Tyr-Ile-Asn-Asn-Trp-Glu-Gln-Ala-Lys DTx 141-157 Gly-Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn-Lys-Thr DTx 224-237 DTx 513-526 Gly-Tyr-Gln-Lys-Thr-Val-Asp-His-Thr-Lys-Val-Asn-Ser-Lys have very high and predefined specificity, and thus provide invaluable tools with which to study the antigenic structure of the protein. We now report the preparation of site-specific antibodies to the two functionally different fragments of DTx, using synthetic peptide conjugates as immunogens. The peptides were chosen from the primary amino acid sequence of DTx previously deduced by Greenfield et al. (1983). Methods Reagents and general procedures. All peptide synthesis reagents and N-a-9-fluorenylmethoxycarbonyl-amino acids were from Novabiochem UK and were of the highest purity available. Pin Technology solid-phase peptide synthesis blocks were from Cambridge Research Biochemicals. HPLC cartridges packed with Spherisorb S50 DS2 were from Chrompack. SDS-PAGE reagents were from National Diagnostics. Nitrocellulose membrane was from Amersham. Immunochemical reagents and reagents for tissue culture were from Sigma except for Auro Dye Forte protein stain which was from Cambio. The reagents necessary for the preparation of maleimide-activated keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) for peptide conjugation were from Pierce, and Freund’s complete and incomplete adjuvants were from Difco. For solid phase immunoassay, 96-well polystyrene immuno-Nunc plates (Life Technologies) were used and all plastic-ware for tissue culture was from Falcon. All other reagents were of the highest grade available. Purified diphtheria toxin J G 1039/10 (5 mg protein ml-l, 1560 Lf ml-I) and J G 1002/10(8 mg ml-l, 2560 Lf ml-l) were provided by Wellcome Biotechnology Research Laboratories, Beckenham, Kent and freeze-dried preparation 79/1 (1000 Lf per ampoule) was from the RIVM, The Netherlands. Choice of peptides, synthesis and coupling to carrier proteins. Peptide selection was based on computer prediction of likelihood of surface location and hydrophilicity, using Genetics Computer Group (GCG) multiple sequence software package (Devereux et al., 1984) and loop formation (Edwards et al., 1991). Some consideration was also given to possible involvement of certain regions in toxin function (Tweten et al., 1985; Rolf et al., 1990). The sequences selected are shown in Table 1. Peptide DTA was custom synthesized by Cambridge Research Biochemicals and the anti-peptide antibody prepared essentially as previously described (Perera & Corbel, 1990). Peptides DTBl and DTB2 were synthesized using a NovaSyn K R polyamide support and N-a-9-fluorenylmethoxycarbonyl amino acids on an LKB Biolynx 4170 peptide synthesizer. Amino acids were coupled as pre-formed symmetrical anhydrides or as pentafluorophenyl esters. For the purpose of conjugation to carrier proteins, cysteine was added to the N- terminus. Cleavage and deprotection was carried out in 13 Mtrifluoroacetic acid in the presence of phenol, 1,Zethanedithiol and thioanisole as cation scavengers. The purity of the peptides was confirmed by reversed phase HPLC using a Spherisorb S5 ODs2 column (100 mm x 3 mm) monitored at 210 nm. The peptides had the expected composition on amino acid analysis, determined as the ophthaldehyde-p-mercaptoethanol derivatives (Jones et al., 1981) and relative molecular masses as determined by fast atom bombardment mass spectrometry (M H+ : m / z DTBl calculated = 1529-79 and m/z DTB2 calculated = 1707-94 and found = 1529.70; found = 1706.90). m - Maleimidobenzoyl- N- hydroxysuccinamide - ester- derivatized KLH or BSA was added at 2mg per 1.3 pmol cysteinyl peptide, determined by the free thiol content of the preparation using Ellman’s reagent (Ellman, 1959). On average, 60-70% of the peptide was conjugated. Null conjugates were also prepared, where cysteine was substituted for the peptide. All conjugates were purified by gel filtration on a Sephadex (G-50) column and stored in phosphate buffered saline (PBS : 1a5 mM-potassium phosphate, 8-1 mM-sodium phosphate, 2.7 mM-potassium chloride, 137 mM-sodium chloride, pH 7.5). Protein concentration was determined by the method of Bradford (1976), using crystalline BSA, fraction V, as standard. + Immunization. Female New Zealand White rabbits (3 kg body weight, from Froxfield Farms Ltd, Petersfield, Hampshire, UK) were immunized with 10C160 pg peptide conjugate in PBS mixed 1 : 1 with Freund’s complete adjuvant, in a total volume of 0.5 ml, by subcutaneous and intradermal injections. One booster injection was administered 14 d later using 50 pg peptide in incomplete adjuvant. Rabbits were killed by cardiac puncture on day 28. Control (preimmune) blood samples were collected from a marginal ear vein of the same rabbits before immunization started. Sera were prepared from clotted blood and stored at -30 “C until required. Purijkation of antibodies. Antisera were purified by affinity chromatography as previously described by Edwards et al. (1989). Immunological analyses. ELISA microtitre plates were coated with 100 p1 peptide (2 pg ml-l), peptide conjugate (1 pg ml-l), purified diphtheria toxin or diphtheria toxoid (5pg ml-l), in coating buffer (0.05 M-carbonate buffer, pH 9.6). Washing steps were performed in PBS/Tween buffer (PBS containing 0.05 % Tween 20), and blocking steps in 1% (w/v) skimmed milk powder (Marvel) in PBS. The detecting antibody was anti-rabbit IgG conjugated to horseradish acid peroxidase with 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic and hydrogen peroxide as co-substrates. A405was determined using an Anthos plate reader, model 2001 (Anthos Labtec Instruments, Austria) with ARCOM 2.4 software running on an Olivetti 286 PC. The binding titre was defined as the concentration of antibody that gave halfmaximal binding to an antigen. Western blotting was performed essentially as described by Towbin et al. (1979) where, on average, 0.5-2.5 pg purified reduced diphtheria toxin was applied to 10% (w/v) SDS-PAGE gels and run in a mini- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 Anti-diphtheria toxin site-directed antibodies 1 1 2 2 1 2 1 2 199 2 DTx B A Aurodye KLH-DTA KLH-DTB 1 DTxd Fig. 1. Western blotting of diphtheria toxin fragments with anti-peptide and anti-toxoid antisera. Purified diphtheria toxin, 2-5 pg (1) or 0.5 pg (2) per lane, was subjected to 10% (w/v) SDS-PAGE and immunoblotted with rabbit antisera to peptide DTA (KLH-DTA), peptide DTBl (KLH-DTB1) or to the whole diphtheria toxoid (DTxd). One nitrocellulose membrane was stained with Auro dye following electrophoretic transfer. A and B indicate the positions of the respective DTx fragments at M , 20000 and 38000 and DTx indicates the same for intact toxin at M , 58000. Table 2 . Binding titre of anti-peptide and anti-diphtheria toxoid antibodies in ELISA The serum titres shown are the dilutions, starting with the neat serum, that gave half-maximal binding. In the case of purified anti-peptide antibody the titre shown is that dilution that gave half-maximal binding, starting from a purified antibody preparation of 100 pg specific IgG ml-l. Binding titre Anti-peptide antisera Antigens DTA DTB2 DTBl Homologous peptide* KLH-peptide* DTx native DTx urea 1 :32 1 :2200 1:800 1:1600 1 : 1000 1 :10000 >1 1:30 1 :6500 1 :40000 1 :3000 1~5600 + Purified Ab DTB 1 1 :2500 1 :10000 1 :2500 Epitope mapping. The General Net Epitope Scanning strategy (Cambridge Research Biochemicals Pin Technology) was used to synthesize simultaneously 172 peptides, each of 8 aa residues, with an overlap of 6, spanning the entire length of DTx fragment B. The peptides were synthesized on the tips of the derivatized polyethylene pins in the configuration of 96-well microtitre plates (Geysen et al., 1987), as detailed in the Epitope Scanning Kit manual provided by the manufacturers. The N-termini of the peptides thus synthesized were acetylated, prior to deprotection with trifluoroacetic acid/anisole/ethanedithiol. Finally, the pins were neutralized by sonication in 0.1% hydrochloric acid in methanol and stored under silica gel as a desiccant. Immunoassay of the peptides on pins was performed by the modification of the ELISA procedure described above. Non-specific binding sites were saturated by overnight incubation of pins with 2% (w/v) BSA in PBS. Following use, the pins were regenerated by sonication as detailed in the Epitope Scanning Kit manual. ND * Results shown are for the orthologous peptide sequence only. None of the antisera reacted with any of the heterologous peptide sequences. ND, not determined. Protean I1 (Bio Rad) electrophoresis cell. The co-substrates for Western blotting were 4-chloro-a-naphthol and hydrogen peroxide. In vivo toxin neutralization assay. Female Dunkin Hartley guinea pigs (450-500 g) from Harlan Olac (Bicester, Oxon, UK) were shaved and injected intradermally (0.2 ml) with mixtures containing diphtheria toxin, test antibody or both. The animals were observed for 48 h. The protective effect of a test antiserum was assessed by its ability to neutralize a dermonecrotic dose of DTx at the site of injection (B. P. Vol I1 Appendix XIV B2). Results Antisera from rabbits immunized with the three different peptides conjugated to KLH all reacted with the respective immunogen with high titre, as well as to the corresponding peptide in ELISA (Table 2). The binding titre of anti-peptide DTBl to the orthologous peptide conjugate was 4-18 fold higher than that of the other two preparations, Following affinity purification, the binding titre of the anti-peptide antibodies for the orthologous immunogen was 1000-fold greater than for the carrier protein. Although there were large differences in Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 2200 D . Sesardic, V . Khan and M . J . Corbel 0.8 0.6 < wl 0.4 0.2 I I I I I I 0 30 60 90 Peptide 120 150 30 60 L 180 1.5 1.2 0.9 < YI 0.6 0.3 90 Peptide 120 150 180 Fig. 2. Reactivity of antisera to peptide DTBl (a) and DTB2 (b) to multiple peptides encompassing the whole of DTx fragment B. A modified ELISA procedure was used to screen the reactivity of two of the anti-peptide antisera, a-DTB1 (a) and a-DTB2 (b), against 172 overlapping octapeptides, spanning the whole of DTx fragment B. Peptides were synthesized using Pin Technology (Cambridge Research Biochemicals) as described in Methods. the binding titre of the antibodies to the peptides alone, this could reflect differences in the ability of the peptides to bind to the microtitre plate. Of the three anti-peptide antibodies prepared, two also reacted strongly with native DTx, in ELISA. The antipeptide DTBl antibody had the highest titre of binding Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 Anti-diphtheria toxin site-directed antibodies 1.5 1.2 0.9 8 pl 0.6 0.3 ** *t 9 t 0.8 (b1 0.6 < 0.4 0.2 a y* y ** $I * *9 & > c a > id c D * ** ** $ ** ** * 2 Fig. 3. Mapping of epitope for anti-DTB1 (a) and anti-DTB2 (b) antisera. Only the regions of maximum response from Fig. 2a and 2b are shown for reactivity of anti-DTB1 (a) and anti-DTB2 (b) antisera against peptides of DTx fragment B. One letter symbols represent amino acids contained in the peptide to which the antiserum was originally produced and * denotes other amino acids in the sequence. to peptide, conjugate and to the toxin. None of the antipeptide antibodies showed a reduction in binding titre following denaturation of the DTx with 8M-urea. Indeed, the antiserum to peptide DTB2 bound to DTx only after such denaturation of the antigen (Table 2). The 220 1 antiserum to DTA bound much better to DTx, both native and denatured, than did the anti-DTB2 antiserum. Immunoblotting of purified, reduced diphtheria toxin with the anti-peptide antibodies showed that the antisera were highly selective for the corresponding toxin fragment. In contrast, a rabbit antiserum to DTxd reacted with both fragments (Fig. 1.). Antiserum to peptide DTB2 also blotted only to DTx fragment B, but at a lower dilution than for antiserum to peptide DTBl (data not shown). The antiserum to peptide DTBl (and to peptide DTA) bound better to DTx than to DTxd in ELISA, using three different preparations of DTx and six DTxds. Whereas there was no significant difference between the binding of a polyclonal DTxd antiserum to DTx or to any of the DTxd preparations, the antiserum to peptide DTB 1 bound significantly (p < 0.001) better to DTx (average of 60-fold)than to the DTxd preparations (data not shown). The results of epitope mapping of antisera to peptides DTBl and DTB2 on to the DTx fragment B sequence indicated that each of the anti-peptide antisera is highly specific to a cluster of 4-5 different peptides (Fig. 2a, b), from the total of 172 analysed. The antiserum to synthetic peptide DTBl bound to 8 peptides, with maximum binding to two with the sequences MSESPNKT and ESPNKTVS (Fig. 3a), from which it can be deduced that the epitope for this antiserum is predominantly in the C-terminal part of the peptide, sequence SESPNK. Similarly, the antiserum to peptide DTB2 bound only to 4 peptides, with maximum binding to one containing the sequence KTVDHTKV (Fig. 3 b). It is therefore deduced that the epitope for this antiserum is localized to the sequence KTVDH, which occurs towards the middle of the peptide. The affinity-purified antibodies were tested for their ability to protect against DTx challenge in the guinea pig intradermal test in vivo. Rabbit polyclonal antiserum to DTxd was used as a positive control. None of the antipeptide antibodies was protective when used singly or in combination (data not shown). Discussion Site-directed antibodies with high reactivity and specificity against the two functionally distinct fragments of DTx (A and B) have been prepared. These antibodies reacted specifically with the fragment containing the sequence against which they were raised, confirming the utility of the approach in generating fragment-specific antibodies. Often the most suitable region against which to direct an anti-peptide antibody is an amino acid sequence Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 2202 D . Sesardic, V . Khan and M . J . Corbel predicted to occur in a loop region (i.e. a linear sequence of residues), with a high hydrophilicity index, i.e. most likely to occur at the surface of the protein. Such regions are often very antigenic, and an antibody against such a region should recognize both native and denatured protein, as well as the immunizing peptide. A suitable region on DTx fragment B was identified on this basis, peptide DTB 1, and the anti-peptide antibody produced did indeed have the highest binding titre to the native toxin, amongst the anti-peptide antibodies obtained. This is consistent with the fact that DTBl has the highest surface and loop likelihood indices of the three peptides. The ability of this antibody to bind to the toxin as well as to the immunizing peptide, and the similarity between the reactivity profiles of the antiserum with native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop region and that the structure of the peptide is similar to the conformation of this region in the native protein, i.e. a linear epitope. This prediction was supported by epitope mapping using overlapping peptides to fragment B. Anti-peptide DTB 1 antibody reacted only with 6 C-terminal aa of the 14-mer immunizing peptide (cys-gpiknmSESPNKt). This 6 aa sequence has the highest probability of forming a loop. As the peptide was coupled to carrier protein via its Nterminal cys, these C-terminal residues, with a high probability of forming a surface loop, would be most accessible for antibody generation, and would be highly accessible for antibody-binding in both the native and denatured toxin. The location of the predicted epitope for anti-DTB1 antibody, to occur in a surface loop region of DTx, was recently confirmed by X-ray crystallography (Choe et al., 1992). While the anti-peptide DTB2 antibody bound well to the immunizing peptide, this antibody reacted only weakly with the toxin, and then only when it was denatured with 8 M-Urea, suggesting that the epitope for this antibody is not readily accessible in the toxin, possibly because the immunizing peptide adopts a conformation different from that of the corresponding region of the protein. Some evidence that this might be so was obtained by epitope mapping. Although it is possible, and perhaps even likely, that a mixture of antibodies is present in the polyclonal anti-peptide antiserum, most appear to be directed against a narrow region within the peptide, the 5 aa situated centrally in the 14-mer (cys-gpyqKTVDHtkvnsk). However, it is the C-terminal 6 aa residues that have a high surface prediction. Hence, the epitope that is most antigenic in the peptide is not the most accessible in the toxin. This is consistent with the X-ray crystallographic structure of DTx, in which the predicted epitope for anti-DTB2 forms part of a P-sheet adjacent to a short surface loop/turn region (Choe et al., 1992). The anti-peptide DTA antibody bound well to the toxin, both native and denatured. Interestingly, the Nterminal 6 aa of DTA have a high probability of forming part of a surface loop sequence. Whilst the epitope for this antibody has not been mapped, the ability of the antibody to bind to both native and denatured toxin suggests that the dominant immunogenic epitope on the peptide is accessible and contiguous within the toxin. Inspection of the X-ray crystallographic structure of DTx (Choe et al., 1992) reveals that, in fact, peptide DTA includes two loop regions, one at each end of the sequence. Binding of the anti-peptide antibodies to DTx was significantly greater, 60-fold in the case of anti-DTB 1, than to various preparations of DTxd, presumably reflecting variable modification of the epitopes during toxoid preparation. It is of interest that an anti-toxoid antiserum did not recognize any of the synthetic peptides. Such toxin-selective antibodies could be of considerable value in monitoring the loss of specific surface epitopes during toxoiding. One of the primary objectives of raising site-specific antibodies against toxins is to obtain reagents that affect toxin function, particularly by neutralization. However, none of the anti-peptide antibodies reported here protected against DTx challenge, in an in vivo intradermal challenge test in guinea pigs. This indicates that these regions (more specifically their epitopes) do not represent protective epitopes, although it is still possible that they form part of such an epitope, which is nonlinear. Clearly, studies such as these will help to delineate the structure-function relationships of DTx, particularly when allied with recent information on the X-ray crystallographic structure of the toxin. The uniqueness of the deduced epitopes for the antipeptide DTBl and DTB2 antisera was determined by searching the PIR protein database (release 28.0, March 1991) using the GCG sequence analysis (Devereux et al., 1984) for matching sequences. No sequence identical with the anti-DB1 epitope was found, whereas two sequences, in a hypothetical protein of yeast, identical with the anti-DTB2 epitope were found amongst a total of 28,232 sequences analysed. If a single mismatch was allowed in the matching algorithm, then 20 sequences were identified for the anti-DTBl epitope and more than 200 for the anti-DTB2 epitope. This demonstrates the unique specificity of the antiserum to peptide DTBl for DTx fragment B. We thank Mr P. A. Knight and Dr A. Whitaker of Wellcome Research Laboratories for supplying the diphtheria toxin used in this work. We are grateful to Dr P. H. Corran (Division of Chemistry, NIBSC) and Dr R. Wait (Division of Pathology, Centre for Applied Microbiology and Research, Porton) for performance of HPLC, amino acid analyses and mass spectrometric analyses on the peptides, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33 Anti-diphtheria toxin site-directed antibodies respectively. We are also indebted to Dr R. J. Edwards of the Department of Clinical Pharmacology, RPMS, London for his help with predictions for the presence of loop regions within the diphtheria toxin. References AUDIBERT, F., JOLIVET, M., CHEDID,L., ALOUF,J. E., BOQUET,P., RIVAILLE, P. & SIFFERT, 0. (1981). Active antitoxic immunization by a diphtheria toxin synthetic oligopeptide. Nature, London 289, 593-594. AUDIBERT, F., JOLIVET, M., CHEDID,L., ARNON,R. & SELA,M. (1982). 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Reassembled HVJ (Sendai virus) envelopes containing non-toxic mutant proteins of diphtheria toxin show toxicity to mouse L cell. Nature, London 266, 839-840. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:45:33