REFERENCES. Aebersold, R. H., Teplow, D. B., Hood, L. E. & Kent, S. B. H. .... cliffe, J. G. (1990) Production and characterization of monoclo- nal antibodies to ...
Eur. J. Biochem. 219, 161-169 (1994) 0 FEBS 1994
Antagonism of the intracellular action of botulinum neurotoxin type A with monoclonal antibodies that map to light-chain epitopes Isabelle CENCI DI BELLO', Bernard POULAIN', Clifford C. SHONE3, Ladislav TAUC' and J. Oliver DOLLY' ' Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, England * Laboratoire de Neurobiologie Cellulaire et MolCculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France Public Health Laboratory Service, Porton Down, England (Received July 23/0ctober 6, 1993) - EJB 93 1110/3
mAbs were produced in mice against highly purified, renatured light chain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on nitrocellulose to avoid the undesirable use of toxoids. Subcutaneous implants of relatively high amounts (up to 10 pg each) of LC allowed its slow release into the systemic circulation and, thus, yielded much higher antibody titres against the underivatized antigen than had hitherto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG, and IgG,, subclasses reactive specifically with BoNT A and LC, in native and denatured states, without showing any crossreactivity with types B, E, F or tetanus toxin. The pronounced reactivities of three mAbs towards refolded LC or intact toxin, observed in immunobinding and precipitation assays, relative to that seen in Western blots imply a preference for conformational epitopes. Though mAbs 4, 5 and 7 failed to neutralize the lethality of BoNT in vivo,administration intraneurally of mAb7 prevented the inhibition of transmitter release normally induced by subsequent extracellular administration of BoNT A. Notably, the latter mAb reacted with a synthetic peptide corresponding to amino acids 28-53 in the N-terminus of the LC, a highly conserved region in Clostridial neurotoxins reported to be essential for maintaining the tertiary structure of the chain. Most importantly, when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transiently, the blockade of acetylcholine release by the toxin; this novel finding is discussed in relation to the nature of the zinc-dependent protease activity of the toxin.
Botulinum neurotoxin A (BoNT A) is one of seven serologically distinguishable types of structurally related proteins produced by the anaerobic bacterium, Clostridium botuhum. These neurotoxins (reviewed in Dolly et al., 1988, 1990, 1992; Dolly, 1992) have an approximate M, of 150000 and consist of a light chain (LC; M, = 50000) disulphide linked to a heavy chain (HC; M, = 100000) when proteolytically activated. This family of highly toxic proteins blocks, specifically and with high potency, the release of acetylcholine (ACh) from peripheral nerve terminals by a triphasic mechanism that involves binding, internalization and intracellular action (Simpson, 1989; Dolly, 1992). At mammalian motor-nerve endings, the disulphide-linked dichain form of BoNT seems necessary for productive uptake (de Paiva et al., 1993a); removal of the C-terminal region of the HC results in loss of acceptor binding (Poulain et al., 1989a,b; Dolly et al., 1992). In Aplysia neurons, the N-terminal half was shown to contain a domain involved in translocation of the LC (Poulain et al., 1989a,b). LC alone blocks Ca2+Correspondence to Prof. J. 0. Dolly, Department of Biochemistry,Imperial College of Science, Technology and Medicine, South
Kensington, London, England SW7 2AY Fax: +44 71 225 0960. Abbreviarions. BoNT A, botulinum neurotoxin type A ; LC, light chain; HC, heavy chain ; PEG, poly(ethy1ene glycol) ; i.p., intra-peritoneal; ACh, acetylcholine; LD,,, amount of toxin that killed half the animals within 4 days.
dependent release of transmitters when delivered inside murine motor nerves by means of liposomal entrapment (de Paiva and Dolly, 1990) or added to digitonin-permeabilised chromaffin cells (Bittner et al., 1989; Stecher et al., 1989) or PC-12 cells (McInnes' and Dolly, 1990). In contrast, in Aplysia, the additional intra-neuronal presence of HC (Poulain et al., 1988, 1989b) is necessary for the action of the LC. Availability of antibodies directed against functional domains of the LC would be invaluable for probing its intracellular action in these different systems and for pinpointing essential residues. An array of polyclonal and mAbs to several BoNT types has been produced which recognise LC or HC epitopes in the parent toxin only (Shone et al., 1985; Gibson et al., 1987; Simpson et al., 1990); some cross-reacted with other types (Oguma et a]., 1982; Hambleton et al., 1984; Kozaki et al., 1986, 1989; Tsuzuki et al., 1988), indicating their sharing of common antigenic determinants. However, anti-BoNT available to date have all been raised against toxoid preparations; most of the resultant antibodies exhibiting significant toxinneutralization activity are directed to HC epitopes (Kozaki et al., 1989) whereas only one LC-specific antibody has shown weak neutralizing ability (Kozaki et al., 1989; Simpson et al., 1990). Thus, in the present study, animals were immunized with renatured LC to maximize the success of obtaining mAbs that would be directed against the active-site regions and, consequently, be more useful to probe the intracel-
162 lular mechanism of action of the toxin. This yielded a panel of mAbs with different properties, including two that antagonised the intra-neuronal blockade of acetylcholine release by BoNT; an epitope for one of the latter was pinpointed to the N-terminus of the LC, confirming the location therein of an essential domain (Kurazono et al., 1992).
EXPERIMENTAL PROCEDURES Materials The NSO myeloma cell line was obtained from the European Collection of Animal Cell Cultures, Public Health Laboratory Service, Porton Down, Wiltshire, England. NalZ5-I was purchased from ICN. Poly(ethy1ene glycol) 4000 (PEG), myoclone plus foetal calf serum and Dulbeccos’s modified Eagle medium were from Gibco, whilst the antibody isotyping kit was obtained from The Binding Site. Goat anti(mouse IgG) - alkaline-phosphatase conjugate was purchased from Bio-Rad Laboratories. Immulon IV ELISA plates were from Dynatech. All other reagents were obtained from Sigma Chemical Co. The sea-hare species, Aplysia californica, was purchased from Marinus Inc.
subcloning. Ascitic fluids were produced in BALBlc primed with Freund’s incomplete adjuvant.
Synthesis of LC peptides Peptides were synthesized using an automated, solidphase peptide synthesiser (model 431 A, Applied Biosystem Inc.) using Applied Biosystem FastMocTMchemistry and 4 - (2’,4’- dimethoxyphenyl -N- (9- fluorenylmethoxycarbonyl) aminoethyl) phenoxy resin (Novabiochem Ltd). The purity of the peptides was assessed by reverse-phase HPLC.
ELISA Titres were obtained by coating the test plates overnight with 7 pmol/well (= 1 pg BoNT A or = 0.33 pg LC/well) or with a fivefold molar excess of LC peptides. Hybridoma supernatants or ascitic fluids were added at appropriate dilutions and the assay, thereafter, using alkaline-phosphatase conjugated secondary antibody and p-nitrophenyl phosphate substrate carried out as recommended by the manufacturer. Non-immune sera and a mAb raised against an unrelated protein were used as negative controls whilst mouse polyclonal sera from hyperimmunised animals, whose spleen were utilised in the fusion procedures, served as positive controls.
Preparation and purity of the immunogen LC of BoNT A was separated as described by Maisey et al. (1988). An additional step, on a Mono-Q-Sepharose chromatographic column, to remove any contamination by unnicked toxin was included to decrease the toxicity of the resultant LC. Its purity was ascertained by SDS/PAGE under denaturing conditions and by mouse toxicity (Maisey et al., 1988). Protein was revealed by silver staining or electrotransferred to nitrocellulose membrane and detection with rabbit polyclonal antibodies, prepared in this laboratory, that react specifically with the LC of BoNT A; the latter method was used in preference to the mouse bioassay to test the purity of the antigen under preparation.
Immunisation protocol Primary doses of immunogen were administered into adult BALB/c mice by subcutaneous implantations (on the dorsal aspect of the neck) of = 5 pg LC immobilised on a nitrocellulose membrane. This was followed by 4 -6 intraperitoneal (i.p.) injections of LC (= 6-8 pghnjection) mixed 1 : 1 with incomplete Freund’s adjuvant at 10-14-day intervals. In two (out of six) animals, a secondary subcutaneous nitrocellulose implant (= 10-12 pg) was inserted in the hindleg and given in lieu of the multiple i.p. boosters. On reaching adequate antibody titres (1 : 5000 to 1: 10000, monitored by ELISA and dot-immunobinding assays), final daily intravenous (= 3 pg) and i.p. doses (= 5 pg) without adjuvant were given in the 4 days immediately preceeding the fusion.
Fusion procedure Spleens from two hyper-immunised BALB/c mice and myeloma NSO-1 cells were fused using a modification of the standard PEG method (Newel1 et al., 1988), with minor modifications according to Orlik and Altaner (1988) and Muniz et al. (1992). Antibody-secreting hybridomas were identified by dot-immunobinding assay and ELISA prior to
Western-blot analysis BoNT A samples (0.1-0.5 pg) were subjected to SDS/ PAGE under reducing conditions in 8% gels and transferred to nitrocellulose membranes according to Aebersold et al. (1986). To demonstrate serotype and chain specificities, blots were probed with hybridoma supernatants or with ascitic fluids at appropriately selected dilutions. Immuno-staining was carried as for ELISA but developed with bromochloroindolyl phosphate/nitroblue tetrazolium substrate.
Dot immunobinding assay Ascitic fluids or hybridoma supernatants were screened for their reactivities by application to nitrocellulose membranes; = 1 pmol toxin samples, an identical amount known to adsorb in ELISA (calculated by the ratio of bound to unbound radiolabelled lZ5Itoxin in the wells) were applied to the membrane, in 20 mM Tris/HCl, 150 mM NaC1, pH 7.4 (buffer A), in a dot-blot apparatus. Thereafter, the assay was performed as for ELISA and Western-blot analysis.
Immunoprecipitation assay BoNT A was routinely radiolabelled with lzsI to high levels of specific activity (600-1250 Ci/mmol) using a modification of the chloramine-T method (Williams et al., 1983) and LC and HC separated chromatographically (Maisey et al., 1988). 12sI-labelledBoNT A or its chains (15000-50000 cpm; = 5-15 fmol) were mixed with ascitic fluids, serially diluted in buffer A containing 1% bovine serum albumin (to a final volume of 200 pl) and incubated overnight at 4°C. The antibody concentration of diluted samples was kept constant by inclusion of non-immune sera. Goat anti-(mouse IgG) coupled to agarose beads (30 pl suspensiodassay) was added and incubated with mixing at 4 “C for a further 4-5 h. Samples were sedimented and the pellets washed with the above incubation buffer containing 0.05% (by vol.) Tween, prior to quantitation in a y counter. Deter-
163 minations were carried out in triplicate and the results presented are from at least three separate experiments.
Measurement of neutralising abilities of mAbs in vivo and in vitro For these studies, 2-100 times the 50% mouse lethal dose of BoNT A (LD50)were mixed with selected purified mAb IgG (2-4 mg/ml) and incubated with the toxin overnight prior to i.p. administration to mice. The remaining BoNT A toxicity was determined using the mouse time-death method (Boroff et al., 1966) as the LD,, assay could not be applied for the range of toxin dilutions administered. Rabbit polyclonal anti-LC IgG, mouse antisera (from mice immunised with LC and used in‘fusions) and pre-immune sera were used as positive controls and negative controls, respectively. Antagonistic effects of the antibodies on the inhibition of neurotransmitter release were assessed in invertebrate Aplysia neurons, as detailed by Poulain et al. (1989b).
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RESULTS Purity of BoNT A LC used for immunization and preparation of hybridomas Refolded LC rather than neurotoxoid was used as immunogen with the aim of obtaining a library of mAbs having unique specificities that might afford protection against the intracellular action of the toxin. The purity of the LC preparation was established by the presence of a single band revealed in Western blots by rabbit polyclonal anti-LC antibodies. An additional chromatographic step on Mono-Q Sepharose was necessary to remove degradation products that were detectable by immunoblotting but not by silver staining. This apparently also removed minute traces of single-chain BoNT because the toxicity of the resultant LC was decreased substantially (from 4 X lo3 to 2X lo2 mouse LDJ mg). In initial attempts at producing mAbs, mice were injected with 1-2 pg purified LC, but this resulted in death of some animals ; those surviving became intoxicated by subsequent secondary i.p. boosters of antigen in amounts exceeding 2 pg. In later experiments, this difficulty was overcome by administering subcutaneous implantations of LC (=5 pg) immobilised on nitrocellulose membrane. Although animals primed in this way could be boosted by i.p. injections of not more than 8 pg LC, a single booster (after 2 months) with a 10-12-pg implant produced much higher (2-3-fold) antibody titres than those found in mice immunised repeatedly, at fortnightly intervals, by i.p. injections amounting to more than 30 pg immunogen. This slow delivery of nitrocellulosebound immunogen over a 2-3-month period and the final multiple doses of antigen (Ratcliffe et al., 1990) administered on four successive days prior to fusion, together with the added conditioning of the cells to low concentrations of PEG, were all contributory factors to achieving high antibody yields. Hybridomas were observed in about 25% of the 968 seeded wells ; 23 clones exhibiting the highest level of secreted antibodies were selected by ELISA. Although single-clone hybrids were produced in primary cultures, further subcloning by limiting and serial dilution was performed to ensure monoclonality. Seven stable hybridoma cell lines, which retained high secreting activity in the later stages of cloning, were ultimately generated. Isotype analysis indicated that six of the clones secreted mAbs of IgG, subclass except for mAb 7 which was of the IgG,, subclass (Table 1).
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-[log(ascitic fluid dilution)]/foid Fig. 1. ELISA titres of mAbs against BoNT and LC. Plates were coated with the specified antigen, BoNT (a), LC (b) (7 pmol/well equivalent to 1 pg BoNT and 0.33 Fg LC, respectively) and mAb reactivities determined as indicated under Experimental Procedures. Titres were derived from these curves and expressed as the dilution of individual mAb A that produces an absorbance, greater or equal to ( 2 ) 1 and are summarised in Table 1 . Symbols for mAbs are 2, 0 ; 3, V ; 4, V ;5, 0 ; 6, U; 7 , A. indicated as 1 , 0;
Significantly higher amounts of ascitic fluids were produced by injecting these secreting hybridomas into animals primed with Freund’s incomplete adjuvant (Gillette, 1987) rather than the conventional pristane.
Specificities of the anti-LC mAbs Reactivity in ELISA and dot blot imrnunobinding assays Interaction of each mAb with BoNT A, its LC and HC, and BoNT subtypes B, E, F and tetanus toxin, a related Clostridial protein, was analysed by ELISA. No reactivity was observed with any of the above-mentioned types (not shown) except for BoNT A and its LC (summarised in Table 1). The ELISA titres were derived from curves, represented graphically in Fig. 1, and are expressed in a more interpretable form (Table 1) as the dilution of antibody that produced an absorbance 2 1. Control assays using a mAb against an unrelated antigen or pre-immune sera produced absorbances of < 0.5 at 10-fold dilution. From Fig. l a , mAb 2 is seen to be the most reactive with BoNT A because the negative log of thc
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Fig.2. Exclusive reactivity of mAbs for BoNT type A. Samples of BoNT subtypes A, B, E, F and TeTX in replicate 8% gels were subjected to SDSPAGE under reducing conditions. Constituents HC and LC of each type of neurotoxin were visualised by silver staining of the gel (a) and in the corresponding Western-blotted gel, immunostained by incubation with each mAb (c). As shown for the representative mAb 3 (b), reactivities are limited to LC A and intact BoNT A (c). Relative reactivities (dilutions) of the mAbs are indicated in Table 1 .
dilution that gives an absorbance 21 corresponds to an apparent 5 X lo5 dilution; equivalent values for more weakly interacting mAbs (nos 6 and 7) ranged over 1- 2.5 X 104-fold. Some of the mAbs (nos 1-3 and 7) exhibited the same spectrum of reactivities with similar or fractionally higher titres when LC rather than BoNT was bound to the plates (Fig. 1b and Table 1). In contrast, results obtained for mAbs nos 4 -6 showed much lower titres (lo2) with LC than BoNT, suggesting that the antigen may be bound to the wells in a different orientation and, thus, be less reactive with these particular antibodies. Due to the weak reactivity of some mAbs in ELISA, an additional dot immunobinding assay was selected to ensure that these would not be lost due to an inability to detect their different specificities. Optimal
165 0 mAb 7
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Fig. 3. Immunoprecipitation of "5-I-labelled BoNT A. BoNT A was radiolabelled to high specific activity and 5-15 fmol 'Y-iabelled toxin mixed with the appropriately diluted mAb, incubated overnight and immune complexes sedimented with anti-mouse IgG coupled to agarose beads (as described under Experimental Procedures). The total antibody concentration was kept constant by inclusion of the requisite amount of non-immune sera. Results are expressed as the maximum amount of T - B o N T associated with the pellets. In all cases, the values obtained for control samples, containing an irrelevant mAb, have been subtracted from the totals to yield the amounts which are specifically precipitated by each mAb. The immunoprecipitation of '251-BoNTand lZ5I-LC(not graphically represented) are summarised in Table 1. Mab 1, A; 2, 3, 4, 0; 5 , X ; 6, 0; 7, A.ppt, precipitate.
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Fig. 4. Intra-neuronaly applied mAb 7 prevents the inhibitory action of BoNT A applied via the bath. ACh release was evoked by an action potential in the two specified pre-synaptic neurons in the buccal ganglion of Aplysia; amplitudes of ensuing responses were recorded against time (see text). After control recordings were made, each neuron was injected (arrows) with either mAb 7 ascitic fluid (0) or a control mAb (H) raised against an unrelated protein; then BoNT A (hatched area) was applied at a concentration of 10 nM to the extracellular medium. Note the ensuing depression of ACh release recorded from the cell injected with control antibody compared to the sustained uninhibited neurotransmission in the other member of the pair of neurons that received mAb 7.
Exclusive reactivity of mAbs for BoNT A
stained denatured LC at dilutions ranging approximately over lo2- 103-fold, whilst dilutions in excess of lo4were insufficient to reduce the prominent immunoreactivity observed with mAbs 2 and 7 (Table 1). Staining of the LC by mAb 6 was the weakest in intensity even at lo2 dilution, but was nevertheless detectable relative to control blots probed with a mAb against an unrelated antigen or pre-immune sera that lacked any noticeable staining (not shown). In general, poor immunoreactivity demonstrated by Western blot was also reflected by low ELISA titres (mAbs 1>4 > 6) against LC bound to the plates, whereas the dot-immunoblot assay of those same mAbs consistently showed enhanced sensitivity, presumably due to differences in the mechanisms of antigen immobilization. The other mAbs (2, 3 and 7) gave similar high titres in all assays, suggesting that their epitopes remained exposed after coating to the solid support.
In order to ascertain the immunological cross-reactivity of the mAbs towards different serotypes of BoNT, samples (in replicate gels) of types A, B, E, F and TeTX were separated by SDSPAGE under reducing conditions. One gel was silver stained to identify the constituent denatured HC and LC of each toxin type (Fig. 2a); the others were transferred to nitrocellulose and exposed to individual mAb ascitic fluids (Fig. 2b,c). The major immunoreactive product detected by all mAbs was an intense band corresponding to LC of BoNT A. With all of the mAbs, there was a total absence of crossreactivity towards HC of type A or for either chain of other BoNT types and tetanus, as shown using mAb 3 (Fig. 2b). Mabs 2 and 7 were particularly sensitive in detecting less than nanogram amounts of unnicked single-chain BoNT A, just visible by silver staining (Fig. 2a). The reactivity of mAbs varied markedly. For example, mAbs 1, 4 and 5
Immunoprecipitation of 1251-labelled BoNT A and LC mAbs that interacted with apparent high affinity on Western blot and ELISA, especially those showing optimum reactivities in dot-immunobinding assays, were then tested for their ability to immunoprecipitate radioiodinated BoNT A or LC. As this methodology measures mAbs binding to native antigen in solution, it could select antibodies with different properties, thereby complementing the data obtained by the other assays. The toxin was labelled by the chloramine-T technique and LC separated according to stringent procedures established in this laboratory (Williams et d., 1983) and known to conserve the biological activity of BoNT. Maximal levels of precipitation of specifically bound '2SI-BoNT and '25-I-LC were obtained for the following mAbs (4 > 5 > 6; Fig. 3 and Table 1). Other mAbs exhibited very
amounts of either BoNT or LC (1 pmol/dot) were applied to nitrocellulose membranes and the limit of immunodetection for each mAb determined (Table 1). In general, the recognition patterns shown by the mAbs in the dot-immunobinding assays were 2- 100-fold more sensitive than the ELISA method; in particular, the immunoreactivity of mAbs 4 and 5 towards LC bound to nitrocellulose was enhanced 2500fold. The implication from this study is that antigen adsorbed to polystyrene plates could have assumed a different conformation to that presented by LC or BoNT bound to nitrocellulose and illustrates that the use of different assays is important in detecting mAbs with a wide range of specificities.
166
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Time (hours) Fig. 5. Blockade of transmitter release induced by BoNT A is reversed by injection of anti-LC mAbs into Aplysia neurons. (A) After BoNT A had been applied via the bath (hatched area) [lo nM, (0)or 30 nM (M)] and 50% of the blockade of neurotransmission ensued, mAb 7 ascitic fluid (M) or purified mAb 7 IgG (0)(final concentration 200 nM) was injected into pre-synaptic neurons. This was followed by the recovery of ACh release. (B) BoNT A was applied via the bath at 25 nM (hatched area), then purified mAb 7 IgG was injected (arrows) into the pre-synaptic neurons at different times (80 min or 160 min) after application of BoNT A. The intracellular IgG concentration (final) at either time was 170 nM. The extent of recovery of ACh release depended on the time of injection; mAb injected at 80 min, after toxin application produced virtually full recovery but a much lower restoration of activity resulted from injection at the later time of 160 min; recovery was not sustained and transiency was noticeable in experiments performed for longer than 4 h. (C, D) After BoNT A (10 nM) induced a significant blockade of ACh release, mAb 4 ascitic fluid (C) or control ascites (0)(D) or a mAb raised to an unrelated protein (M) were injected into pre-synaptic neurons; recovery of neurotransmission was seen only in the neuron injected with mAb 4. None of the antibodies exerted any effect on neurotransmission in non-toxin treated neurons.
poor abilities to immunoprecipitate even at low dilutions (10'). The important characteristic revealed by this data is that mAbs 4 and 5, in particular, and mAb 6 to a lesser degree, interact strongly and specifically with BoNT or LC in their native state. The other mAbs reacted feebly with the latter but yet exhibited high titres when tested against antigen coated on a solid support, suggesting that recognition is dependent on a particular conformation and/or antigen concentration.
The epitope of mAb 7 maps to a peptide in the N-terminal sequence of LC In an attempt to identify the epitopes recognised by the mAbs, their reactivities were screened against two synthetic peptides corresponding to sequences of BoNT A LC, namely residues Gly28-Asn53, and Ah23 -Asn227 (Thompson et al., 1990). Only mAb 7 reacted strongly and specifically with the N-terminal peptide; a fivefold molar excess of peptide relative to the amount of BoNT A usually used, produced an equivalent titre in ELISA (5X104) and dot blot (lo5;Table 1) to that observed with BoNT A or renatured LC. Furthermore, the latter mAb showed no reactivity towards another synthetic N-terminal peptide directed to the first 16 residues of the chain, unlike mAbs 1 and 3 which interacted feebly with this peptide, producing absorbances of < 0.5 at dilutions of 200-fold. Rabbit or mouse polyclonal sera against LC, which were used as positive controls, exhibited immunoreac-
tivity towards all three peptides but at dilutions of < l o 2 fold.
Two mAbs neutralize the intracellular action of BoNT A in Aplysia Absence of effects in mice in vivo
From the above findings, mAbs 4 and 7 were selected for their ability to diminish the toxicity of BoNT in vivo and in vitro because the former gave maximal immunoprecipitation of '"I-BoNT, and the epitope of the latter was mapped to the N-terminal region of the LC, a possible functional domain. For these studies, each mAb was incubated with BoNT A and subsequently administered to mice i.p. Both mAbs proved ineffective in modifying the biological activity of the toxin; the onset of symptoms and the time of death were unchanged relative to those shown by mice which had been injected with toxin alone. This may be attributable to the avidity of these antibodies being low relative to the high potency of BoNT. In contrast, control polyclonal anti-LC IgG purified from rabbit sera neutralized 2.5 X lo6 mouse LD,, unitdm1 serum. Similarly, the same mAbs failed to antagonise the neuromuscular blocking activity of BoNT A when their effects were evaluated using the mouse phrenic nerve hemidiaphragm preparation (de Paiva and Dolly, 1990).
167
Intra-neuronally applied mAb 7 prevents the inhibitory action of BoNT A applied via the incubation bath To test the antagonistic effects of the antibodies on BoNT A-induced blockade of evoked neurotransmitter release, identified cholinergic couples of cholinergic neurons from the buccal ganglion of Aplysia were used because easy access can be gained by intracellular injection. In the first series of experiments, abilities of mAbs to protect neurons against a subsequent application of toxin were tested. mAb 7 ascitic fluid was injected into a presynaptic neuron of the buccal ganglion without alteration of ACh release (Fig. 4); after a delay of approximately 1 h, to allow for migration of antibody towards the nearby nerve terminals (distance of 300500 pm), BoNT A (10 nM) was added to the bath but there was no change in the amplitude of evoked post-synaptic responses ( n = 3, Fig. 4), as normally produced following such an exposure to toxin. This neutralizing effect was sustained for at least the two subsequent hours, indicating that the injected mAb was able to prevent the inhibitory effects of BoNT A. Importantly, the release of ACh evoked from the second pre-synaptic neuron afferent to this post-synaptic cell, from the same preparation, was depressed by the presence of BoNT A; this was to be expected because it had only been injected with a control, irrelevant mAb.
Recovery of BoNT-induced blockade of neurotransnzission by injection of mAbs 4 and 7 Having established that the intracellular application of mAb 7 could prevent the blockade of evoked ACh release induced by BoNT A, the ability of this mAb to arrest the intoxication process was tested. When BoNT A (10nM or 25 nM) was applied via the bath and 50% of the blockade of neurotransmission had ensued, injection of mAb 7 ascites ( n = 4) or purified IgG ( n = 6) into the pre-synaptic neuron produced a recovery of neurotransmission and the depressed responses were restored to 90% of their initial amplitude (Fig. 5A). However, the extent of recovery depended on the time that had elapsed between the application of toxin and the mAb injection (Fig. 5B), indicative that only part of the toxin had been sequestered by the antibody. A calculation of the actual intracellular concentration of mAb 7 within cells, based on injecting 1% of the total cell volume and assuming an homogenous distribution of mAb, yielded an estimate of a final concentration of 170-200 nM; this produced 80% recovery of neurotransmission when 25 nM BoNT A was applied extracellularly (Fig. 5B). Injection of mAb 4 also antagonised the BoNT-induced blockade but the observed recovery of neurotransmission was slower (Fig. 5 C). Notably, no such alleviation of the intoxication was seen when mAbs raised against unrelated proteins were used (Fig. 5D), indicating that restoration of neurotransmission did not result from a non-specific modification of the trigger efficacy or other side effects.
DISCUSSION The properties of antibodies raised to highly purified, renatured BoNT LC rather than to neurotoxoids have not been described until now. Both polyclonal and monoclonal antibodies resulting from immunisation with the detoxified BoNTs were shown to react with HC and LC antigenic determinants but an insufficient number were LC-specific mAbs and generally did not afford protection against the biological
activity of the toxin (Kozaki et al., 1986; Simpson et al., 1990). Our approach was to produce mAbs in response to LC immobilised on nitrocellulose, the rationale being that mAbs raised, for the first time, to a renatured antigen would interact with native LC and, hopefully, neutralize the intracellular action of BoNT, a goal actually achieved. The immunisation employed generated seven mAbs, selectively reactive with BoNT A and its LC. Their differing sensitivities in ELISA and dot irnmunobinding were attributed to distinct mechanisms of antigen immobilization between the two systems, variations which have been noted by others (Brennand et al., 1986; Overall et al., 1989). In addition, the ability of mAbs 4, 5 and 6 to bind native antigen in solution was reflected by their effectiveness at immunoprecipitating '"I-labelled BoNT or LC. All mAbs were exclusively specific for BoNT A and did not cross react with other B, E and F types or TeTX. This suggests that these mAbs, selected for their recognition of BoNT A LC epitopes, do not share common antigenic determinants with any other serologically distinguishable types, a finding reported by others (Kozaki et al., 1986; Gibson et al., 1987). Recently, an alignment of the individual LC sequences of five BoNT serotypes (Kurazono et al., 1992) with that of TeTX revealed that, despite their similar action, these neurotoxins overall exhibit low sequence similarity, with the most conserved segments being located in the central core region of the LC. It is of great interest, thus, to have mapped mAb 7 to a defined N-terminal peptide sequence (Gly28Asn53), a region deemed essential in maintaining the conformation of the chain (Kurazono et al., 1992). Although the degree of sequence identity and similarity is greatest ( ~ 5 2 % ) between LC of BoNT B and TeTX (BoNT A similarity =32%), the number of identical or related residues that align in this N-terminal portion of the individual LC of the toxin is very high (Kurazono et al., 1992). It was shown recently (Schiavo et al., 1992a) that the core region of TeTX and BoNT includes a zinc-binding motif characteristic of neutral metalloproteases and that it houses the catalytic site by which CZostridiaE toxins proteolytically cleave their target (Schiavo et al., 1992a,b). The target of this activity for both TeTX and BoNT B has been demonstrated both in vitro and in vivo to be a synaptic-vesicle-associated membrane protein, VAMP IT (Link et al., 1992; Schiavo et al., 1992b; Poulain et al., 1993) also known as synaptobrevin (Baumert et al., 1989). The use of specific inhibitors of metallo-endoproteases has indicated that BoNT A is also a protease (de Paiva et al., 1993 b) . An important outcome of our present study is that it gives some insights into the properties of the target for BoNT A. Indeed, the fact that it is possible to reverse the intoxication process, even transiently, clearly indicates that the intraneuronal substrate for BoNT A has either a high turnover rate or that it also exists in a form which is not directly accessible for proteolysis (but becomes susceptible eventually), thus forming a reservoir of target because, under some conditions, near complete recovery of neurotransmission was seen even when release had been reduced by BoNT A to less than 70% of the control level. For example, only certain pools of vesicles, from the several that have been documented, are readily releasable (Prado et al., 1992). A BoNT-susceptible protein with a high turnover being implicated in the mechanism is unlikely as treatment of ApZysia synapses with the proteinsynthesis inhibitor, cycloheximide, was without effect on neurotransmitter release (Mochida et al., 1990). In the context of the results presented here, if recovery was due to
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(1992) in Handbook of Phnrmacology (Herken, H. & Hucho, F., eds) pp. 681 -717, Springer-Verlag, Berlin. Gibson, A., Modi, N. K., Roberts, T. A. & Shone, C. C. (1987) Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system, J. Appl. Bact. 63, 217-226. Gillette, R. W. (1987) Alternatives to pristane priming for ascitic fluid and monoclonal antibody production, J. Immunol. Methods 99, 21-23. Hambleton, P., Shone, C., Wilton-Smith, P. & Melling, J. (1984) in Bucterial protein toxins, pp. 449-450, Academic Press, London. Kozaki, S., Kamata, T., Takahashi, M., Shimizu, T. & Sakaguchi, G. (1986) The use of monoclonal antibodies to analyze the strucA number of peptides to BoNT-A light chain were synthesized ture of Clostridium botulinum type E derivative toxin. Infect. and gifted by Dr Schmidt of the US Army Medical Research in Immun. 52,786-791. 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