Non-toxic Fragment Research Articles

In vivo molecular imaging for a nasal botulism vaccine in mice and nonhuman primates (53.8)

Abstract Nasal administration is an effective route for a needle-free vaccine. However, safety concerns were raised by the potential of nasally administered antigens to reach the central nervous system. We tested the real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a recombinant nontoxic fragment of botulinus toxin A (BoHc/A), by using a newly established in vivo imaging method, [18F]-labeled BoHc/A-positron emission tomography (PET). This method provides results that are consistent with direct counting of [18F] radioactivity or the traditional [111In]-radiolabel method in dissected tissues of mice and non-human primates. In addition, no deposition of BoHc/A in the cerebrum and olfactory bulb was found after nasal administration of [18F]-BoHc/A in mice and non-human primates. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in non-human primates. These results demonstrate a new way to use in vivo imaging for safety evaluation of nasal vaccines.

Chitosan‐based gene delivery vectors targeted to the peripheral nervous system

A non-toxic, targeted, simple and efficient system that can specifically transfect peripheral sensorial neurons can pave the way towards the development of new therapeutics for the treatment of peripheral neuropathies. In this study chitosan (CH), a biodegradable polymer, was used as the starting material in the design of a multicomponent vector targeted to the peripheral nervous system (PNS). Polycation-DNA complexes were optimized using imidazole- and thiol-grafted CH (CHimiSH), in order to increase transfection efficiency and allow the formation of ligand conjugated nanocomplexes, respectively. The 50 kDa non-toxic fragment from the tetanus toxin (HC), shown to interact specifically with peripheral neurons and undergo retrograde transport, was grafted to the binary complex via a bi-functional poly(ethylene glycol) (HC-PEG) reactive for the thiol moieties present in the complex surface. The targeting of the developed nanocomplexes was assessed by means of internalization and transfection studies in the ND7/23 (neuronal) vs. NIH 3T3 (fibroblast) cell lines. Targeted transfection was further confirmed in dorsal root ganglion dissociated primary cultures. A versatile, multi-component nanoparticle system that successfully targets and transfects neuronal cell lines, as well as dorsal root ganglia (DRG) primary neuron cultures was obtained for the 1.0 (w/w) HC-PEG/DNA formulation.

572 Unprecedented results of a clinical validation of the dedicated and highest resolution breast PET

The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC).Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model.We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment.CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells.C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules.These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium.

Targeted gene delivery into peripheral sensorial neurons mediated by self-assembled vectors composed of poly(ethylene imine) and tetanus toxin fragment c

A simple, safe and efficient system that can specifically transfect peripheral sensorial neurons can bring new answers to address peripheral neuropathies. A multi-component non-viral gene delivery vector targeted to peripheral nervous system cells was developed, using poly(ethylene imine) (PEI) as starting material. A binary DNA/polymer complex based on thiolated PEI (PEISH) was optimized, considering complex size and zeta potential and the ability to transfect a sensorial neuron cell line (ND7/23). The 50 kDa non-toxic fragment from tetanus toxin (HC), which has been previously shown to interact specifically with peripheral neurons and to undergo retrograde transport, was grafted to the complex core via a bifunctional PEG (HC-PEG) reactive for the thiol moieties present in the complex surface. Several formulations of HC-PEG ternary complexes were tested for targeting, by assessing the extent of cellular internalization and levels of transfection, in both the ND7/23 and NIH 3 T3 (fibroblast) cell lines. Targeted gene transfer to the neuronal cell line was observed for the complex formulations containing 5 and 7.5 μg of HC-PEG. Finally, our results demonstrate that the developed ternary vectors are able to transfect primary cultures of dorsal root ganglion dissociated neurons in a targeted manner and elicit the expression of a relevant neurotrophic factor.

Transcutaneous delivery of tetanus toxin Hc fragment induces superior tetanus toxin neutralizing antibody response compared to tetanus toxoid

Transcutaneous immunisation is a promising vaccination delivery strategy which targets potent immune cells residing in the outer layer of the skin. In this study, the immunogenicity and neutralizing potency of the non-toxic Hc fragment of tetanus toxin (HcWT) and a mutant of Hc lacking ganglioside binding activity were compared with that of tetanus toxoid (TTxd) following transcutaneous immunisation (TCI) of mice. Mice immunised with HcWT in the absence of an adjuvant induced highest anti-toxoid and anti-Hc antibody titres, with a significant increase in the toxin neutralising antibody response compared with TTxd. These results are in contrast to previous studies employing subcutaneous delivery, where TTxd was found to be a more potent immunogen than the Hc fragment of the toxin. We conclude that the HcWT protein is more immunogenic than TTxd when given via the transcutaneous route. Our results suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens which harbour protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunisation.

A Targeted Therapeutic Rescues Botulinum‐A Poisoned Neurons

Botulinum neurotoxin (BoNT) is the most poisonous poison known to mankind for which currently no antidote is available to rescue poisoned nerves. An effective medical countermeasure strategy will be required and would include its efficient delivery specifically to presynaptic nerve terminals. Here we present the report of rescue of botulinum poisoned nerve cells by Mastoparan‐7, a peptide constituent of bee venom that was delivered through a drug delivery vehicle (DDV) constructed from the non‐toxic fragment of botulinum neurotoxin itself. We found that mastoparan‐7 that was delivered into spinal cord cells rescued BoNT/A poisoned cells by restoring over 40% neurotransmitter release. The rescue of the cell poisoning does not seem to occur from inhibition of the endopeptidase activity of BoNT/A against its well known substrate, SNAP‐25, mechanistically involved in the exocytosis process. Rather, Mastoparan‐7 induces a physiological host response apparently unrelated to SNAP‐25 but linked to the phospholipase signal transduction pathway. Our results open new avenues to examine mechanism of exocytosis process, and also to examine alternative cellular mechanism of botulinum neurotoxin action. Successful demonstration of BoNT‐based DDV opens door for its utility for drug delivery against many neuronal and neuron‐muscular disorders.This work was supported JSTO‐CBD/DTRA #3.10003_05_WR_B

A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons

AbstractBotulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerve cells by Mastoparan-7 (Mas-7), a peptide constituent of bee venom, that was delivered through a drug delivery vehicle (DDV) constructed from the non-toxic fragment of botulinum neurotoxin itself. We found that Mas-7 that was delivered into BoNT/A intoxicated cultured mouse spinal cord cells restored over 40% of stimulated neurotransmitter release. The rescue of the cell poisoning did not occur from inhibition of the endopeptidase activity of BoNT/A against its well known substrate, SNAP-25 that is mechanistically involved in the exocytosis process. Rather, Mas-7 induced a physiological host response apparently unrelated to SNAP-25, but linked to the phospholipase signal transduction pathway. In addition to providing the first effective antidote against botulism, our results open new avenues to study the mechanism of exocytosis, and also to examine an alternative cellular mechanism of botulinum neurotoxin action. An effective BoNT-based DDV can also be utilized for drug delivery against many neuronal and neuromuscular disorders.

Recombinant AttenuatedSalmonella entericaSerovar Typhimurium Expressing the Carboxy-Terminal Domain of Alpha Toxin fromClostridium perfringensInduces Protective Responses against Necrotic Enteritis in Chickens

Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain chi8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10(9) CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.

Breaking up the correlation between efficacy and toxicity for nonviral gene delivery

Nonviral nucleic acid delivery to cells and tissues is considered a standard tool in life science research. However, although an ideal delivery system should have high efficacy and minimal toxicity, existing materials fall short, most of them being either too toxic or little effective. We hypothesized that disulfide cross-linked low-molecular-weight (MW) linear poly(ethylene imine) (MW<4.6 kDa) would overcome this limitation. Investigations with these materials revealed that the extracellular high MW provided outstandingly high transfection efficacies (up to 69.62+/-4.18% in HEK cells). We confirmed that the intracellular reductive degradation produced mainly nontoxic fragments (cell survival 98.69+/-4.79%). When we compared the polymers in >1,400 individual experiments to seven commercial transfection reagents in seven different cell lines, we found highly superior transfection efficacies and substantially lower toxicities. This renders reductive degradation a highly promising tool for the design of new transfection materials.

One dose or two? The use of polymers in drug delivery

Controlled-release delivery systems are designed to prolong the release of drugs within the body. They consist of drug molecules encapsulated within biodegradable polymers which degrade safely into small, non-toxic fragments, resulting in the release of the drug. Because different polymers have different degradation times, the system can be tailored to achieve the desired release rate and this can be very useful in treatments where daily dosing is required, such as epilepsy. Despite this obvious advantage, there are problems with this type of delivery, including an increased risk of overdosing. This article examines the pros and cons of controlled-release drug-delivery systems. Copyright © 2007 Society of Chemical Industry

Rapid degradation of gliadin peptides toxic for coeliac disease patients by proteases from germinating cereals

We report the isolation and characterisation of proteases from germinated wheat, rye and barley, and their ability to degrade gliadin peptides toxic for coeliac patients. It is shown for the first time that these proteases cleave these peptides rapidly into non-toxic fragments with less than nine amino acids. These proteases have distinct advantages when compared to bacterial or fungal proteases, and are promising candidates for the detoxification of gluten containing foods and for oral therapy for celiac patients.

Expression of diphtheria toxin inStreptococcus mutansand induction of toxin-neutralizing antisera

The nontoxic full-length diphtheria toxin (DTX), fragment A (DTA), and fragment B (DTB) were each genetically fused to the major surface protein antigen P1 (SpaP) of Streptococcus mutans. Repeated attempts to express the recombinant DTX and DTB in the live oral vaccine candidate Streptococcus gordonii were unsuccessful, whereas DTA could be readily expressed in this bacterium. However, the recombinant DTX, DTB, and DTA could be expressed in the related oral bacterium S. mutans. Western blotting and enzyme-linked immunosorbant assay (ELISA) using anti-DTX and anti-P1 antibodies demonstrated the expression of the three fusion proteins in S. mutans. Mouse antisera raised against the recombinant S. mutans recognized the native DTX in Western immunoblotting. The antibodies raised against S. mutans expressing the recombinant DTX and DTA neutralized the cytotoxicity of the native toxin in a Vero cell assay, but the neutralization titers were relatively low. The potential of using S. gordonii as a live vaccine against diphtheria faces major challenges in the expression of DTX in this organism and in the induction of high-titer toxin-neutralizing antibodies.

Pathophysiology of psoriasis: coping endotoxins with bile acid therapy

The authors have tested the hypothesis that the deficiency of bile acids and the consequent endotoxin translocation might play a role in the pathogenesis of psoriasis. Under normal conditions the bile acids act as detergents (physico-chemical defense) and can protect the body against enteric endotoxins by splitting them into nontoxic fragments and thus preventing the consequent release of cytokines [Persp. Biol. Med. 21 (1977) 70]. A total of 800 psoriasis patients participated in the study and 551 were treated with oral bile acid (dehydrocholic acid) supplementation for 1–8 weeks. The efficacy of the treatment was evaluated clinically and also by means of the Psoriasis Area Severity Index (PASI score). During this treatment, 434 patients (78.8%) became asymptomatic. Of 249 psoriatics receiving the conventional therapy, only 62 (24.9%) showed clinical recovery during the same period of time ( P<0.05). The curative effect of bile acid supplementation was more pronounced in the acute form of psoriasis (95.1% of the patients became asymptomatic). Two years later, 319 out of the 551 acute and chronic psoriasis patients treated with bile acid (57.9%) were asymptomatic, compared to only 15 out of the 249 patients (6.0%) receiving the conventional treatment ( P<0.05). At the end of the 2-year follow-up, only 10 out of 139 acute psoriasis patients (7.2%) receiving the conventional therapy and 147 out of 184 bile acid treated patients (79.9%) were asymptomatic ( P<0.01). To conclude, the results obtained suggest that psoriasis can be treated with success by oral bile acid supplementation presumably affecting the microflora and endotoxins released and their uptake in the gut.

Surface decontamination of simulated chemical warfare agents using a nonequilibrium plasma with off-gas monitoring

InnovaTek, Inc., Richland, WA, is developing a surface decontamination technology that utilizes active species generated in a nonequilibrium corona plasma. The plasma technology was tested against dimethyl-methyl phosphonate (DMMP), a simulant for the chemical agent Sarin. Gas chromatograph mass spectrometry analysis showed that a greater than four log/sub 10/ destruction of the DMMP on an aluminum surface was achieved in a 10-min treatment. An ion-trap mass spectrometer was utilized to collect time-resolved data on the treatment off-gases. These data indicate that only nontoxic fragments of the broken down DMMP molecule were present in the gas phase. The technology is being further refined to develop a product that will not only decontaminate surfaces but that will also sense when decontamination is complete.

Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice.

The function of the nervous system is a consequence of the intricate synaptic connectivity of its neurons. Our understanding of these highly complex networks has profited enormously from methods used over the past two decades that are based on the mechanical injection of tracer molecules into brain regions. We have developed a genetic system for the mapping of synaptic connections during development of the mammalian central nervous system and in the mature brain. It is based on the transsynaptic transfer of green fluorescent protein (GFP) in the brains of mice using a fusion protein with a nontoxic fragment of tetanus toxin (TTC) expressed in defined neurons. These transgenic mice allowed us to visualize neurons, at single-cell resolution, that are in synaptic contact by the detection of GFP in interconnected circuits. Targeted genetic expression with a specific promoter permitted us to transfer GFP to defined subsets of neurons and brain regions. GFP-TTC is coexpressed with a lacZ reporter gene to discriminate neurons that produce the tracer from cells that have acquired it transneuronally. The marker shows selective transfer in the retrograde direction. We have used electron microscopic detection of GFP to define the ultrastructural features of the system. Our work opens up a range of possibilities for brain slice and in vivo studies taking advantage of the fluorescence of GFP. We point the way toward the use of powerful multiphoton technology and set the stage for the transsynaptic transfer of other proteins in the brains of mice.

Use of a rationally attenuated Bordetella bronchiseptica as a live mucosal vaccine and vector for heterologous antigens

Inactivation of the aroA gene of Bordetella bronchiseptica severely impaired its ability to colonise the respiratory tract of mice. The B. bronchiseptica aroA mutant was investigated as a live vaccine and vector for heterologous antigens. The B. bronchiseptica aroA mutant expressing the non-toxic fragment C (FrgC) of tetanus toxin (strain GVB120) was used to immunise mice intranasally. Immunised mice produced a strong serum and mucosal antibody response to B. bronchiseptica and serum anti-FrgC antibodies. Upon challenge with wild type B. bronchiseptica, immunised mice rapidly reduced the numbers of B. bronchiseptica in their respiratory tract, although clearance was more pronounced in the lower than in the upper respiratory tract. Immunisation with GVB120 protected ∼40% of mice from tetanus toxin challenge. As far as we are aware, this is the first description of a recombinant B. bronchiseptica strain being used as a live vaccine vector for heterologous antigens.

Tetanus toxin modulates serotonin transport in rat-brain neuronal cultures.

As has been previously described, tetanus toxin (TeTx) and its H(C) fragment inhibit the sodium-dependent 5-hydroxytryptamine (5-HT) uptake in rat-brain synaptosomes, probably through a kinase mechanism affecting the 5-HT transporter. Now, the inhibition of 5-HT uptake in neurons in primary culture by TeTx in a dose-dependent manner is described in this work. This effect is also produced by the nontoxic C-terminal fragment of the TeTx heavy chain (H(C)-fragment), indicating that 5-HT uptake inhibition is a consequence of the toxin binding to the plasmatic membrane and not to its catalytic activity. This conclusion is supported by the fact that the 5-HT accumulation was not inhibited by the light chain of TeTx or the toxoid, and was even potentiated by botulinum neurotoxin A. These results correlate with the activation of phosphoinositide-phospholipase C activity in the cultures used in this study, this activity only being enhanced by TeTx and by its Hc-fragment. On the other hand, the use of tyrosine phosphorylation modulators indicates that both Na3VO4 and basic fibroblast growth factor (bFGF) produce an enhancement of 5-HT uptake in this system, which is also sensitive to TeTx inhibition. On the other hand, genistein alone is able to reduce the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests that TeTx and genistein may share some events in their respective mechanisms of action. Furthermore, the incubation at different concentrations of 12-O-tetradecanoylphorbol 13-acetate (TPA) confirms the involvement of protein kinase C (PKC) in 5-HT transport modulation in rat-brain neuronal primary cultures. In summary, we shall demonstrate in this work that TeTx induces, through its Hc fragment, an inhibition of both basal and stimulated serotonin uptakes in primary neuronal cultures, in parallel to the activation of phosphoinositide-phospholipase C activity and PKC activation.

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.

Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin.

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.

Identifying the principal protective antigenic determinants of type A botulinum neurotoxin

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT A (H C; residues 861–1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT A-H C were isolated and cloned. Specific binding of the mAbs to BoNT A-H C was demonstrated by surface plasmon resonance, with K ds in the range of 10 −10 to 10 −11 M. These antibodies recognized a genetically engineered polypeptide (1150–1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin H C fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer H C-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the H C-peptides had high levels of antibodies that recognized BoNT A-H C . However, immunizations with only one of the H C peptides protected when mice were challenged with BoNT A . On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.