Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519–845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519–845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519–593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain.

Preparation and characterization of anti-HN519-845 antibodies. Pooled immune serum isolated by immunizing mice with H N 519-845 provided a high antibody titer of 1/64,000 against BoNT/A (Fig. 3a). The Abs were specific to BoNT/A as indicated by absence of Abs binding to BoNT/B. Highly purified Abs (2 mg/ml) were obtained after protein G purification of 1 ml mice sera (Fig. 3b), which showed specific binding to the BoNT/A H-chain (Fig. 3c).
The epitope specificity of Abs were profiled using a solid-phase enzyme-linked immunosorbent assay (ELISA) assay using synthetic overlapping peptides (19 amino acid long with 5 amino acid overlapping regions) spanning H N 519-845 region. Antibody responses were observed for peptides representing C and N-terminal regions of H N 519-845 with high response against N6, N21, N22, N23, and N26, whereas, a moderate to low Ab response against N7, N8, N9, N25, N27 and N28 (Fig. 3d). Inhibition analysis using mouse anti-H N 512-845 Abs showed that the Abs inhibited more than 50% of BoNT/A-SNP interaction (Fig. 3e), indicating the presence of blocking Abs.
Mouse protection assay using anti-HN 519-845 antibodies. Active and passive MPAs were carried out to evaluate the protective efficacy of anti-H N Abs developed by immunizing the mice with H N 519-845 region of BoNT/A. Active MPA showed that the immunized mice were partially protected (75% and 25%) at 10 and 25 × LD 100 doses of BoNT/A with increase in the survival time at 100 and 500 × LD 100 doses of BoNT/A (Fig. 5a). Passive MPAs were carried out using 1/5 and 1/10 dilutions of anti-H N 519-845 sera. Anti-H N 519-845 exhibited 100% protection against 1.05 × LD 100 doses of BoNT/A at 1/5 dilution and 40% protection at 1/10 dilution showing that anti-H N 519-845 can confer protection passively against active BoNT/A (Fig. 5b). showed that a high antibody response was obtained against the N-terminal region (H N 519-593) and C-terminal region (H N 729-845) of H N 519-845 (Fig. 3d). To analyze the regional contribution in development of blocking Abs, H N 519-593 and H N 729-845 were expressed and purified separately (Fig. 6a) followed by Ab generation in mice. High Ab titers were obtained against BoNT/A for both H N 519-593 and H N 729-845 immunized mice (Fig. 6b). Anti-H N 729-845 was profiled using synthetic peptides and the response was compared with Abs generated against H N 519-845 and active BoNT/A. Almost The assays were carried out in triplicates (n = 3 ± SD) (b) Binding of peptide H N 519-845 to SV lysate was determined by adding different concentrations of H N 519-845 (250-3.9 nM) to a SV-coated plate (10 μ g/ml) and incubating the plate at 37 °C for 1 h. The plate was washed and the SV-H N interaction was probed with mouse anti-his antibody followed by using horseradish peroxidase (HRP)labelled anti-mouse antibody. An increase in the binding of H N 519-845 was observed with its increasing concentration, finally becoming saturated at 250 nM concentration (n = 3 ± SD).

Generation of H
comparable profiles were obtained for anti-H N 519-593, anti-H N 729-845 and anti-H N 519-845 representing high immunogenicity of these representative BoNT/A H N regions (Table 1).

Electrophysiology analysis to investigate inhibition of channel formation by anti-H N antibodies.
Application of BoNT/A (4.16 μ g/ml) resulted in channel formation in murine neuro 2a neuroblastoma cells (Fig. 7a). Channel formation was measured by patch-clamp electrophysiology in which the cells were held at − 80 mV (Fig. 7a(i)). Outward currents were observed at positive voltages from 40 mV to 100 mV ( Fig. 7a(ii)). Based on the composition of the pipet solution and the bath solution, the outward current is elicited predominantly by the efflux of K + ions. This was confirmed using a non-specific K + inhibitor TEA (1 mM) which blocked around 90% of the current (Fig. S1B).
Baseline current (control) was subtracted from BoNT/A-mediated channel opening to eliminate the contribution of endogenous neuro 2a currents (Fig. 7a(iii)). A known inhibitor of BoNT/A, toosendanin (TSN), was used as a positive control to prevent channel formation in neuro 2a cells by BoNT/A at 40 μ M (Fig. 7a(iv and v)). Inhibition of BoNT/A-mediated channel formation by Abs against H N 519-845, H N 729-845 or H N 519-593 was tested by incubating BoNT/A with Abs for 2 h at room temperature in The binding was detected using HRP-labelled anti-mouse antibody. A mixture of peptides from the light chain region of BoNT/A was used as negative control. A high antibody response was observed against N6, N7 N21, N22, N23, and N26, whereas, a moderate to low antibody response was observed for peptides N8, N9, N25, N27 and N28. (e) An inhibition assay was carried out using anti-H N 512-845 to check if the pAb can inhibit the binding of 125 I-labelled BoNT/A to SNPs. A fixed concentration of 125 I-labelled BoNT/A (50,000 cpm) was incubated (1 h) with different dilutions anti-H N 512-845 pAb (1/100 to 1/25,600) and 10 μ g of SNPs were added to the mixture and incubated for an additional 20 min. The assay showed that more than 50% inhibition of BoNT/A binding to the SNPs was obtained (n = 3 ± SD). . The toxin-peptide mixture was injected intravenously into 5 mice along with the controls, which were injected with BoNT/A (1.05 × LD100), without peptide. The mice were kept under observation for six days. The assay showed that premixing H N 519-845 showed complete protection (100%) at 25 μ g dose. The protection shown by H N 519-845 was specific for BoNT/A as no protection was observed against BoNT/B at similar dose (data not shown). Combination of peptides N6, N7, N8 and 26, 27, 28 showed a partial protection 60% and 20%, respectively, indicating these regions may be important in generation of blocking antibodies. (b) MPA studies were repeated for H N 519-845 and peptide combination N6, N7, N8 by using a slightly higher dose of BoNT/A (1.5 × LD 100) to confirm their protective efficacy. A complete protection (100%) was observed for H N 519-845 N6, N7, N8 and a partial protection (40%) was observed for the synthetic peptides N6, N7, N8.  (Fig. 7a(vi)). Incubating BoNT/A with pre-immune sera did not affect BoNT/A-mediated channel formation and served as negative control for the analysis (Fig. 7a(vi)).

Mouse protection assay using anti-H N 519-593 and anti-H N 729-845 antibodies. Passive MPAs
were carried out using H N 519-593 and H N 729-845 specific Abs to determine the protective efficacy of these regions against lethal dose of BoNT/A (1.05 × LD 100 ) BoNT/A. Anti-H N 519-593 Abs showed 60% protection against BoNT/A, whereas, no protection was observed with anti-H N 729-845 Abs against active BoNT/A (Fig. 7b).

Discussion
Earlier vaccination against BoNT relied on formaldehyde-inactivated toxins (toxoids) and consisted of either monovalent (i.e. one) or pentavalent (five) toxoid serotypes. However, formaldehyde-treated toxins have adverse side effects. Determination of the sequences of BoNT genes has permitted construction of recombinant vaccines. Recombinant H C of BoNT/A1, stimulated immune responses that protected mice against intraperitoneal toxin doses of up to 10 6 LD 50 of BoNT/A1 [9][10][11] . The subdomains of the H C domain (H CN and H CC ), and the catalytic L chain, or the L chain linked to the H N domain (LH N ) have been investigated. It was reported that the H CN and H CC domains of H C , when each used alone or as a mixture, stimulated lower protective immunity in mice than the intact H C by itself 12 . Three monoclonal Abs (mAbs) prepared against BoNT/E L chain, H N and H C were reported to inhibit translocation of BoNTs A and E 13 . Mouse mAbs prepared 14 after immunization with toxoid followed by BoNT/A recognized sites on the light chain, H N and H C . A vaccine based on the L chain and the N-terminal half of the H chain (i.e. LH N ) of toxins A and B has been tested 15 . To improve its protective capability, the LH N /A was cross-linked with formaldehyde. One injection of the cross-linked preparation protected mice against 103 × LD 50 of BoNT/A1 and against BoNT/A subtypes A1, A2, and A3. Also, a single dose of LH N /B provided protection in mice against BoNT/B4 (non-proteolytic toxin subtype). However, the L chain by itself, or the L chain linked to the H N domain (LH N ) induced lower protective immunity than H C 16 . So the H C domain seemed to be the smallest fragment for an optimal vaccine design. Intact whole toxins with mutations at the enzyme active site that detoxified the molecule have also been tested in mice as vaccine candidates and found to be effective in providing protective immunity against toxin poisoning 17,18 .
Using overlapping synthetic peptides spanning the entire BoNT/A and BoNT/B molecules, the antigenic sites were mapped 7,8,[19][20][21] with antisera from human volunteers immunized with a pentavalent toxoid containing BoNTs A, B, C, D and E, and with anti-toxoid antisera of horse, mouse and chicken. The sites that bind to mouse brain SNPs 1,22 have also been mapped using overlapping synthetic peptides that covered the entire polypeptide chains of toxins A and B. It was found that regions on the H C as well as regions on the H N domains participate in the binding of the toxin to Abs and to SNPs. Region 785-803 was immunodominant with antisera of all four host species followed, in decreasing order, by regions 547-565, 743-761, 659-677 and much lower, but reproducible, amounts of Abs were bound, by some other H N peptides. A recombinant peptide corresponding to region H N 729-845 (H N 729-845) of BoNT/A was found to bind directly to SNPs and to neuronal cells and to inhibit considerably the binding of BoNT/A to SNPs 2 . Significantly, the free peptide by itself protected mice against a lethal dose (1.05 × LD 100 ) of BoNT/A and this compared well with the protection provided by free recombinant peptide 1163-1296 in the H CC domain 2 , concluding that H N in its free state can bind to the neuronal cells and inhibit toxin binding to the cell and prevent toxicity.
The epitopes of BoNTs A and B were also mapped 8 with Abs from cervical dystonia patients who were treated with, and became immunoresistant to, native BoNT/A or BoNT/B. Anti-BoNT/A Abs recognized regions 785-803 (28 of 28 patients) and 743-761 (9 of 28 patients). Garcia-Rodriguez et al. 23 obtained two mAbs from human volunteers immunized with a pentavalent toxoid similar to that mentioned above 7 . Using molecular evolution methods, they deduced that the mAbs bound to an epitope around residue 757 in the BoNT/A H N domain, which falls within region 743-761 localized by Dolimbek et al. 8 . The two mAbs neutralized BoNTs A and B in vivo when pre-mixed with 200 × LD 50 of toxin and injected intraperitoneally into 10 mice.
The aforementioned studies 8 established that, upon immunization with the intact toxin, regions 785-803 and 743-761 of BoNT/A stimulated protecting Abs in humans and mice. These regions are located on the helical bundle of the H N domain. But it was not known whether immunization with a suitable fragment carrying these regions would induce Abs that would bind to the same epitopes on the intact protein, and neutralize the toxin. Thus, in the present work, we carried out studies to determine the ability of a properly-selected fragment of H N that carries the aforementioned epitopes to stimulate production of protective Abs against the toxin. If immune responses against an H N fragment are found to be protective, then such a fragment, or perhaps active parts of it, could be incorporated into a synthetic vaccine design.
The present study identified the regions of BoNT/A H N domain which are capable of producing blocking Abs that inhibit binding of the toxin to the cells and its toxicity. To achieve this, we cloned a peptide fragment corresponding to residues 519-845 of BoNT/A. This segment represents almost the entire anti-parallel helical bundle of H N . It retained much of its native folding in solution as confirmed by CD measurements indicating that intra-and inter-helical interactions were maintained in the segment's solution folded structure. The radiolabeled peptide bound to SNPs and to SV lysate. Mouse Abs were generated against this fragment without cross-linking with formaldehyde and by a normal immunization procedure without optimizing the adjuvant. The Abs were characterized based on their submolecular specificity towards BoNT/A regions and also for their ability to inhibit BoNT/A binding to SNPs (Fig. 3e). The Abs were highly specific to the H-chain of BoNT/A (Fig. 3a,c), despite sharing a high sequence homology 24 with BoNT/B. Immunodominant epitopes were identified on both N and C-terminal regions of H N 519-845 compelling us to study these regions independently (Fig. 3d). Initially, we studied H N 519-845 by MPA using synthetic peptides spanning the entire segment. The study implied that N-terminal region of H N 519-845, i.e. H N 519-579, may be instrumental in the translocation process along with certain regions on the C-terminal part distributed randomly (Fig. 4a). Our previous study showed that H N 729-845 can compete with BoNT/A and provide protection at low doses of BoNT/A 2 . To further investigate protection by the H N domain, we expressed H N 519-593 and H N 729-845 individually and injected each in the mice to prepare Abs. The peptides exhibited no toxicity and produced Abs in high titers, recognizing both the peptides and BoNT/A. The Abs were compared to Abs against intact BoNT/A and against H N 519-845, to identify the epitope specificity of each anti-peptide Ab (Table 1). Abs against each peptide bound equally well to the respective peptide and to the intact toxin, indicating that these peptides elicited Abs that were primarily directed against a native-like conformation of the BoNT/A (Table 1 and Fig. 6b). Antibodies against peptides H N 519-845 and H N 519-593 significantly inhibited ion channel formation by BoNT/A on neuro 2a cell membrane, whereas Abs against peptide H N 729-845 caused partial (about 50%) inhibition (Fig. 7a(vi)). Inhibition of channel formation can be either due to increased cargo size or limiting receptor availability 25 . During voltage ramp protocol, part of the outward current is contributed by endogenous ion channels in neuro 2a cells. Voltage-gated K + channels such as Kv1.1, Kv1.4, Kv2.1 and some members of TASK channels, TASK1 and TASK2, were described in neuro 2a cells 26,27 . TEA, a non-specific K + inhibitor, blocked the outward current by 90% suggesting most of the BoNT/A-mediated currents and endogenous channels-mediated currents are due to K + efflux. Remainder could be contributed by Cl − influx, dominant in the external solution. Additionally, in a step protocol from − 100 to 70 mV we recorded a peak inward current at − 10 mV similar to Na + current 28 . However, the inward current was not detected in the ramp protocol and did not affect the BoNT/A mediated-outward current. Therefore inhibition of BoNT/A-mediated ion channels by Abs against H N peptide regions represents inhibition of K + efflux.

Figure 7. Evaluation of anti-H N -fragments in inhibiting BoNT/A-induced channel formation and protection. (a) Effect of BoNT/A and its antibodies on channel formation in neuro 2a cells by whole
Electrophysiology data was supported by MPA analysis, where, antibodies against H N 519-845 and H N 519-593 were protective against lethal dose of BoNT/A. On the other hand, Abs against H N 729-845 were not protective. Peptide H N 519-845 contains all the region represented by H N 729-845 but is 210 residues longer than H N 729-845. Consequently, the specificities possessed by these two Ab responses were different as expected ( Table 1). The antigenic sites recognized by the Abs against H N 729-845 were also present on the H N 519-845 but the latter recognized additional antigenic sites within residues 519-593. This indicated that the Ab responses against region 519-593 are important for protection against the whole toxin. This was in fact confirmed by the finding that Abs against H N 519-593 exerted high (60%) protection against 1.05 × LD 100 of BoNT/A. In addition, even when equimolar amounts of free peptides N6, N7, N8 and N9 were premixed with BoNT/A and the toxin-peptides mixture was injected into mice, it protected them against BoNT/A poisoning.
It is concluded that the H N domain carries binding sites to the cell membrane and is capable of generating blocking (protective) Abs against BoNT/A. Antibodies against appropriate parts of H N will block ion channels on the membrane and in the free state inhibit the toxicity of intact toxin in vivo. The present studies did not test different adjuvants or attempt to maximize/optimize the neutralizing Ab response. It is very hard to compare protective activity of anti-H N 519-593 with that of anti-H C Abs. Those studies 10,29-33 measured toxin challenge doses in LD 50 units, whereas the present work used toxin doses in 1.05 × LD 100 units. We strongly believe that a construct comprising the two domains would provide a more efficient vaccine than either alone. Studies are in progress to compare H N 519-593, H C and the combination construct using the same immunization protocol and challenge dose for optimum vaccine design.

Materials and Methods
Ethics statement. The  Positive clones were expressed in BL21(DE3)pLysS E. coli cells and purified from inclusion bodies, under denaturing conditions, using immobilized metal affinity chromatography (IMAC). The isolated protein was refolded and thoroughly buffer exchanged in phosphate buffered saline (PBS) 2 , followed by its qualitative and quantitative analysis by SDS-PAGE and BCA protein assay kit (Thermo Scientific, Rockford, IL).

Synaptosome binding analysis.
Isolation of SNPs from mouse brain was done as originally described 34 with minor modifications 22 . Peptide was labeled with 125 I (Perkin Elmer, Waltham, MA) by the chloramine T method 35 and binding was analyzed as described by Ayyar  amount of 125 I-labeled peptide (50,000 cpm) was added to different concentrations of SNPs (1.25 to 20 μ g/ ml in 100 μ l of Ringer's buffer). After 20 min incubation at 37 °C SNPs were collected and washed three times with Ringer's buffer and the bound radioactivity was measured in an automatic gamma counter (LKB-Wallac, Turku, Finland). The binding results were corrected for non-specific binding by subtracting the binding values of the controls.
Synaptic vesicle binding analysis. Synaptic vesicles were purified from mouse brain as described by Huttner et al. 36 with few modifications. Briefly, brains from 10 mice were homogenized in 10 volumes of sucrose buffer (320 mM sucrose, 5 mM HEPES-KOH (pH 7.6), 1 mM EDTA, protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO) using glass-Teflon homogenizer. The homogenate was centrifuged for 10 min at 1000 × g and the pellet was resuspended in 0.64 M buffered sucrose. The suspension was centrifuged (45,000 × g) and the pellet was resuspended in PBS containing 2% Triton X-100 and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO) and stirred for 30 min on ice. The lysate was clarified by centrifugation (45,000 × g), aliquoted and stored in − 80 °C.
Peptide-SV binding was analyzed by ELISA by serially diluting the peptide in 1% bovine serum Immunoblotting analysis was carried out by resolving BoNT/A (5 μ g) on a SDS-PAGE gel and transferring the protein to a nitrocellulose membrane. The membrane was blocked with 10% blocking grade milk (Bio-Rad, Hercules, CA) and subsequently incubated with 1/10,000 dilution of purified anti-H N Ab for 1.5 h at room temperature. The membrane was washed 3 × 3 with PBST and PBS and BoNT/A-Ab binding was detected with HRP-labelled anti-mouse IgG (Fc-specific) Ab.
Mapping of antibody epitopes using synthetic H N peptides. A MaxiSorp ™ ELISA plate was coated with 100 μ l of 5 μ g/ml synthetic peptides 1 spanning the H N region. The plate was incubated 1 h at 37 °C and blocked with 3% BSA (w/v) in PBS. Hundred μ l of purified anti-H N Abs (diluted 1/1000 in 1% (w/v) BSA) was added to the plate and incubated for 1 h at 37 °C. The interaction of peptide with specific IgG component was detected by incubating the wells with HRP-labelled anti-mouse IgG (Fc-specific) Ab (1/2000 in 1% BSA (w/v) in PBST) followed by detecting the reaction with 100 μ l of TMB-substrate. The analysis was carried out in triplicates for each peptide and a mixture of BoNT/A light chain peptides was used as control in this study.
Determining in vivo protective efficacy of H N peptides. LD 100 of the active BoNT/A preparation was determined in ICR outbred mice, by injecting different doses (5 mice per dose) of the toxin intravenously and intraperitoneally. The mice were observed 3 times a day for six days. The lowest dose at which Scientific RepoRts | 5:15776 | DOi: 10.1038/srep15776 no mice survived was defined as LD 100 . The protective efficacy of the peptides against BoNT/A was then investigated by mouse protection assays (MPAs). Active BoNT/A (1.05 × LD 100 ) was mixed with H N 519-845 (25 μ g) and with various combinations of peptides (5 μ g each), showing Ab titer on H N 519-845 immunizations (N6+N7, N8+N9, N6+N7+N8, N21+N22+N23, N24+N25+N26 and N26+N+N28) right before injection. The mixtures were then injected intravenously (5 mice per dose). A control group received only 1.05 × LD 100 of active BoNT/A without any peptide. The mice were observed 3 times a day for six days. Protective efficacy of the H N 519-845 and peptides N6+N7+N8 was further assessed by administering the peptides with increased lethal dose of BoNT/A (1.5 × LD 100 ).
Determination of in vivo protective efficacy of mouse anti-H N antibodies. Active and passive MPAs were carried out using anti-H N 519-845 Abs to analyze the blocking Abs in the generated repertoire. Active MPA was carried out by immunizing the mice with a 1:1 ratio of 5 μ g of H N 519-845 and FCA intraperitoneally. The mice were boosted 2 times at 2 weeks intervals by 2 μ g of H N 519-845 in FICA. Bleed was collected 10 days after the second boost and serum was harvested and tittered against BoNT/A. Immunized mice were challenged after a week with different doses of active BoNT/A. Unimmunized mice were used as controls for each dose of the toxin.
For passive MPA, sera containing anti-H N Abs (anti-H N 519-845, anti-H N 519-593 and H N 729-845) were mixed with active BoNT/A (1.05 × LD 100 ) at 1:5 and 1:10 dilutions and incubated for 1 h at 37 °C. The serum-toxin mixture was injected intravenously (5 mice per dose) with control group receiving only 1.05 × LD 100 of active BoNT/A. The mice were observed 3 times a day for six days and protective efficacy of anti-H N 519-845 Ab was evaluated by referencing them with the control group.
Patch-clamp analysis. Whole-cell patch-clamp studies were performed using an automated Port-a-patch setup (Nanion, North Brunswick, NJ) connected to a HEKA EPC 10 USB amplifier as described previously 37 . Patch chips had a resistance of 2-4 MΩ with internal solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 at pH 7.2 and 290 mOsm. The external solution contained 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES at pH 7.2 and 300 mOsm. A voltage ramp protocol was applied to the cells in whole-cell configuration by applying − 100 mV to 100 mV for 600 ms while holding the cell at − 80 mV. The external solution was exchanged for test solutions containing the toxin and/or Abs in the bath chamber. Current density was recorded before and after this exchange and plotted in a bar graph. To assess the efficacy of each antibody in reducing channel formation, the current density measured in the presence of BoNT/A alone was compared to that measured in the presence of BoNT/A and each of the different antibodies using multiple Mann-Whitney U-tests.