Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A.

The potential utility of using DNA vaccination to protect mice from the microbial neurotoxin, botulinum toxin type A, was evaluated. A synthetically derived gene encoding a carboxyl-terminal 50 kDa fragment of the toxin was placed in two sites in the DNA inoculation vehicle pCMVint-BL (Vical), one predicted to lead to MHC I processing (pJT-1 construct) and the other to direct MHC II processing (pJT-2 construct). Mice were then inoculated at 3 week intervals with these two constructs and with the vehicle alone and evaluated for protection from botulinum toxin by i.p. challenges with various toxin doses. Protection was observed at about week 10-11 from toxin doses of 25-100 LD(50). Only animals inoculated with pJT-2 exhibited protection. In dose-response experiments, 50 micrograms of DNA was the minimal dose required to elicit a protective response against serotype A, while protection against serotypes B or E was not obtained. With standard ELISA testing, a relationship was observed between the level of protection and the level of ELISA reactive antibody. Our results support the concept that DNA vaccination is a viable methodology to use in cases where protection from toxins is the goal.

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc.

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc. A mixture of peptides that contained T cell and Ab epitopes was effective in both strains in eliciting T cells and Abs that cross-reacted with Hc. This mixture form gave a quicker rise (after two injections) in cross-reactive (with Hc) Ab titer as compared to other peptide mixtures or the individual peptides, and sustained in BALB/c a high Ab titer upon further booster injections. Some of the regions that elicited crossreactive immunity to Hc have sequence similarity to other clostridial toxins, suggesting that one or more of these synthetic peptides might provide cross-protection against those toxins.

Localization of the regions on the C-terminal domain of the heavy chain of botulinum A recognized by T lymphocytes and by antibodies after immunization of mice with pentavalent toxoid.

We have mapped the regions recognized by T and/or B cells (Abs) on the C-terminal domain (Hc) of the heavy chain of botulinum neurotoxin serotype A (BoNT/A) after immunization of two inbred mouse strains with pentavalent toxoid (BoNTs A, B, C, D and E). Using a set of synthetic overlapping peptides, encompassing the entire Hc domain (residues 855-1296), we demonstrated that T cells of Balb/c (H-2d) mice, primed with one injection of toxoid, recognized two major regions within residues 897-915 and 939-957. After multiple inoculations with toxoid, T cells of Balb/c expanded their recognition ability and responded very well to challenge with peptide 1261-1279 and moderately to stimulation with peptide 1149-1167. Unlike Balb/c T cells, those of toxoid-primed SJL (H-2s) mice exhibited a more complex profile and responded to challenge with a large number of overlapping peptides. After one toxoid injection, however, three peptides, 897-915, 939-957/953-971 overlap and 1051-1069, were the most potent T cells stimulators. After three toxoid injections, peptides 897-915 and 1051-1069 remained immunodominant while the third region was shifted upstream to 925-943/939-957 overlap. The immunodominant epitope within peptide 897-915 was recognized exclusively by T cells, since no Abs were detected against this region. The Ab binding profiles of the two mouse strains were quite similar, showing only small quantitative differences. Both, Balb/c and SJL anti-toxoid Abs displayed strong binding mainly to peptide 1177-1195, followed by peptides 869-887/883-901 overlap and 1275-1296. In addition, a significant amount of Balb/c anti-toxoid Abs was bound to peptide 1135-1153. Unlike Balb/c Abs, that interacted weakly with peptides 995-1013 and 1051-1069, the anti-toxoid Abs of SJL mice exhibited strong binding toward both peptides. The results showed that, in a given strain, the regions recognized by anti-toxoid Abs and T cells may coincide or may be uniquely B or T cell determinants.

Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855-1296) of the toxin.

Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum. Recent studies have shown that immunization with a C-terminal fragment [H(C), residues 855-1296] of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity. The present work was carried out in order to map the molecular and cellular immunological recognition of H(C). We have previously described the synthesis of 31 overlapping peptides encompassing the entire H(C)-fragment of BoNT/A. These peptides were employed in this study to localize the continuous regions recognized by T cells and by antibodies (Abs) generated in two mouse strains against H(C). T cells from SJL that had been primed with H(C) gave a strong proliferative response to challenge in vitro with each of the six peptides spanning residues 897-985 and a lower response to peptide 1051- 1069. While H(C)-primed T cells of BALB/c recognized three regions residing within residues 939-957, 1009-1027 and 1135-1153 (strong). Recognition regions by Abs in SJL or BALB/c anti-H(C) antisera essentially overlapped. However, the level of Abs bound to each region differed between the two strains. These common or similar recognition regions by the two strains were: 855-915 (SJL) or 855-901 (BALB/c); 939-957; 967-1013 (BALB/c) or 981-1013 (SJL); 1051-1069; 1079-1111 (BALB/c) or 1093-1125 (SJL); 1177-1195; and 1275-1296. In addition, BALB/c recognized region 1135-1153. Some of these regions show considerable sequence similarity in BoNT types B and E and, therefore, H(C) of these two BoNTs might offer protection against the correlate clostridial toxins.

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species.

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/1205-1223 overlap, 1261-1279 and 1275-1296. In addition, peptides 883-901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 1149-1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amounts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869-887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093-1111, and 1275-1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Expression of a large, nontoxic fragment of botulinum neurotoxin serotype A and its use as an immunogen.

Using the polymerase chain reaction, a large fragment of botulinum toxin was placed in two expression systems, one designed to produce a fusion protein product and another designed to produce only the toxin fragment. Expression of the fragment in the latter system was inconsistent. Expression of the fusion protein was easily measurable by ELISA. Mice were vaccinated with crude fusion protein, then challenged with native toxin. Mice receiving two immunizations were partially protected from up to 1200 LD50, suggesting that this toxin fragment may be a good vaccine candidate to replace the currently used toxoid.

Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli.

A completely synthetic gene encoding fragment C, a approximately 50-kDa fragment, of botulinum neurotoxin serotype A was constructed from oligonucleotides. The gene was expressed in Escherichia coli, and full-sized product was produced as judged by Western blot (immunoblot) analysis. Crude extracts of E. coli expressing the gene were used to vaccinate mice and evaluate their survival against challenge with active toxin. Mice given three subcutaneous vaccinations were protected against an intraperitoneal administration of 10(6) 50% lethal doses (ID50) of serotype A toxin. The same mice survived when challenged with 3 LD50 of botulinum toxin serotype E but died when challenged with 10 LD50 of serotype E or 3 LD50 of serotype B. Purified fragment C was compared with the botulinum toxoid vaccine in a vaccination and challenge study. Fragment C was as efficacious in protecting against challenge with active botulinum neurotoxin serotype A as the toxoid vaccine. This recombinant protein product has many properties that make it a good candidate for human use to protect against botulinum toxin.

Antibodies having markedly different effects on enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins.

The enzymatic and acetylcholine-releasing activities of two presynaptically-acting phospholipase A2 neurotoxins (pseudexin B and scutoxin) were studied in a synaptosomal fraction. Scutoxin (100 nM) induced greater [14C]acetylcholine release than did pseudexin B (100 nM). Both toxins caused fatty acid production in the synaptosomal fraction, although pseudexin B was more active than scutoxin. One monoclonal antibody raised against pseudexin B (#4) had no effect on the enzymatic activity of either pseudexin B or scutoxin. Two other monoclonal antibodies (#3 and #7), also raised against pseudexin B, antagonized the enzymatic activity of pseudexin B and scutoxin. Monoclonal antibody #3 was more effective than #7 in reducing the amount of acetylcholine released by the toxins, whereas #7 was more effective than #3 in reducing fatty acid production. Although antibody #3 caused complete inhibition of phospholipase A2 activity of pseudexin B on purified substrates, it only reduced phospholipase A2 activity by 35% in synaptosomes. These findings support the hypothesis that gross phospholipase A2 activity does not play a role in stimulation of acetylcholine release by the presynaptically-acting phospholipase A2 neurotoxins.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A2 from Vipera russelli venom.

VRV-PL-VIIIa, the most basic phospholipase A2 (PLA2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal anti-serum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antibodies against VRV-PL-VIIIa were isolated. The monoclonal antibodies exhibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross-reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of the antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Reducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals of each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antibodies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingly, two of the four exhibited the lowest affinities of the monoclonal antibody library for VRV-PL-VIIIa in ELISA, while the other two exhibited the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.

Primary sequence determination of the most basic myonecrotic phospholipase A2 from the venom of Vipera russelli.

The most basic phospholipase A2 (PLA2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC). VRV-PL-VIIIa had an apparent mol. wt of 13,000 and was a single polypeptide. The protein was reduced, pyridylethylated and subjected to sequence analysis. The N-terminal amino acid sequence was established up to the 39th residue. Pyridylethylated VRV-PL-VIIIa was digested with endoprotease Glu-C, and several peptides were purified by RP-HPLC; six purified peptides were sequenced. The sequence of the C-terminal was established by sequencing a CNBr-produced peptide purified by RP-HPLC. Several peptides were also generated by digestion with endoprotease Asp-N. Two peptides were sequenced to obtain overlapping regions. The complete structure was deduced from sequences of overlapping peptides and through homology with other group II PLA2 sequences. Sequence homology was greatest with ammodytoxin A: 99 amino acid residues out of 121 occurred in identical positions. Myotoxin III of Bothrops asper showed 73% homology, 89 out of 121 residues. In agreement with the sequence data, polyclonal antiserum against VRV-PL-VIIIa cross-reacted in ELISA with ammodytoxin A and, to a lesser extent, with caudoxin.

Effects of steroids on association of T-2 toxin with mammalian cells.

The effects of steroids on the association of T-2 toxin with cultured cells were evaluated. Preincubating cells with certain steroids led to a time- and concentration-related increase in total T-2-cell association. At maximally effective concentrations, the increase in association was 300-500%. This effect required a preincubation at 37 degrees C for a minimum of 10 min and was completely reversible after 20-30 min. Steroid treatment increased the rate of toxin-cell association and decreased the rate of dissociation. The effect was elicited by progesterone, estradiol, testosterone and diethylstilbestrol, but not by several other steroids tested. Binding of T-2 to isolated ribosomes was not altered by the steroids. We speculated that steroids somehow alter the state of ribosomal aggregation or assembly such that more toxin can bind after entering the cell.

Specific antibodies against the Zn(2+)-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin.

Although tetanus and botulinum A neurotoxins are ineffective in cultured chromaffin cells, they will inhibit carbachol-induced release of noradrenaline provided they gain access to the cytosol either through artificial pores generated in the plasma membrane or by binding to incorporated exogenous gangliosides. The block of exocytosis persists for weeks followed by a slow recovery of cell function. When specific anti-botulinum A toxin antibodies are introduced into cells through pores after manifestation of the block by botulinum A neurotoxin, restoration of exocytotic function is accelerated and fully reestablished within 4 days. The same time course of restoration is seen with anti-tetanus toxin antibodies in cells poisoned by tetanus toxin. Since the light chains of the toxins are enzymatically active, we have introduced polyclonal and monoclonal anti-light chain antibodies into the cytosol. Of all light chain antibodies tested, only those directed against the peptide homologous to the zinc-binding sequence, which is present in both neurotoxins, restored exocytosis regardless of which toxin caused the block. These results indicate that the zinc-binding domain is directly involved in the interaction of the light chains with their substrates and that the toxins have to be present continuously to maintain the block.

Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells.

We have attempted to establish a cell culture model suitable for molecular mechanism of action studies of necrotic phospholipases A2 (PLA2). Three myonecrotic PLA2 were purified, one basic PLA2 from Naja nigricollis venom and two basic PLA2 (VRV-PL-V and VRV-PL-VIIIa) from Vipera russelli venom. The effects of these PLA2 on several established muscle cell lines were evaluated. As judged by light microscopy, some, but not all, cell lines detached from the culture plate in a time- and concentration-related fashion. Naja nigricollis PLA2 was the most potent at eliciting this effect, followed by VRV-PL-V and VRV-PL-VIIIa. The two most sensitive cell lines, 1447 and 1456, were chosen for further study using N. nigricollis PLA2. Cellular protein and nucleic acid syntheses were inhibited by the toxin in a time- and dose-related manner. However, it appeared that most, if not all, of the inhibition was due to toxin-induced reduction of precursor uptake, suggesting effects at the plasma membrane level. The putative membrane effects were specific, in that uptake of calcium, choline or glucose was not inhibited by the toxin. Moreover, treating the cells with toxin failed to significantly increase lactate dehydrogenase release into the medium. Polyclonal antiserum prepared against N. nigricollis basic PLA2 neutralized the toxicity completely with 1456 cells, but only partially with the 1447 cell line. Both the 1447 and 1456 lines appear to be suitable as cell culture models for necrotizing PLA2 molecular mechanism of action studies.

Effects of emetine on the specific association of T-2 toxin with mammalian cells.

The effects of emetine on the association of T-2 toxin with Chinese hamster ovary cells were examined. T-2 toxin-cell association at both 4 degrees C and 37 degrees C was reduced by up to 90% after preincubation of cells with emetine. Emetine-induced reduction in T-2 toxin-cell association was time-, temperature-, and concentration-dependent. A 4-min preincubation with emetine at physiological temperature was required to develop the maximum inhibitory effect. After brief exposures (< or = 5 min), emetine's inhibitory effects on toxin-cell association were reversible. However, after longer exposure periods to emetine (60 min), toxin-cell association was irreversibly blocked. The addition of emetine to cells prebound with toxin resulted in dissociation at a rate 2 to 3 times slower than a competitive chase with nonlabeled toxin. Emetine did not compete directly for T-2 toxin binding to its receptor on isolated, purified, run-off ribosomes. However, the binding of toxin to purified ribosomes prepared from cells preincubated with emetine was markedly reduced. Scatchard analysis indicated that emetine's inhibitory effects on T-2 toxin-cell association were mediated through mixed allosteric and competitive types of inhibition at specific, intracellular, T-2 toxin ribosomal binding sites.

Effect of emetine on T-2 toxin-induced inhibition of protein synthesis in mammalian cells.

Chinese hamster ovary cells were used to examine the effect of emetine upon the toxicity of T-2 toxin and several related trichothecene inhibitors of polypeptide synthesis. Emetine inhibited protein synthesis and T-2 toxin-cell association in a concentration-dependent manner. The dose-response curves for these two effects were nearly identical. Over a narrow concentration range (0.3-3.0 micrograms/ml), emetine's inhibition of protein synthesis was partially reversible, whereas its inhibition of toxin-cell association was maintained for extended periods. This sustained inhibition of toxin-cell association, resulted in "desensitized" cells with reduced sensitivity to the inhibitory effects of T-2 toxin on protein synthesis. Similar results were obtained when emetine-preincubated cells were challenged with diacetoxyscirpenol, verrucarin A and roridin A. In contrast, there were no measurable effects of emetine upon the response of the cells to the less potent trichothecenes, deoxynivalenol, T-2 tetraol and verrucarol. In addition to emetine, several other inhibitors of polypeptide synthesis were examined for their effects on T-2 toxin-cell association and sensitivity to T-2 toxin. Of these, only cycloheximide inhibited toxin-cell association. Unlike emetine, sustained protection against the effects of T-2 toxin was not observed with cycloheximide.

Binding of myotoxin a to cultured muscle cells.

The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined. Binding was saturable with a moderate binding affinity. Scatchard analysis and Hill plots indicated a single class of binding sites. The binding was reversible, as demonstrated by chase experiments. Radiolabeled myotoxin a bound to the cell surface at a site inaccessible to the general protease, pronase. Specificity and biological relevance of the binding was suggested by competition with unlabeled toxin and various peptides derived from the toxin. Biologically active peptides, corresponding to the N- and C-terminal sequence of myotoxin a, competed with radiolabeled toxin for L8 binding. It was concluded that the L8 system is a suitable cell model to study myotoxin a mechanism of action.

Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects.

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies.

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A2 (PLA2S) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA2. Antibodies neutralizing lethal or enzymatic effects of PLA2 did not recognize linear epitopes.