A Cell Line for Detection of Botulinum Neurotoxin Type B

Abstract

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD₅₀ bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

Keywords: 3Rs; Myobloc; SiMa neuroblastoma; VAMP; botulinum neurotoxin type B; botulinum neurotoxin-sensitive cell line; luciferase; tetanus.

FIGURE 1

BoNT/A-sensitive SiMa neuroblastoma cell line expresses neuronal gangliosides and synaptotagmin but not BoNT/B substrate VAMP2. (A) Three neuroblastoma cells (SiMa, SH-SY5Y and IMR-32) express the BoNT/A substrate SNAP25 but not the BoNT/B target VAMP2. Only the N2A neuroblastoma cell line expresses both SNAP25 and VAMP2. (B) BoNT/B does not cleave VAMP2 in N2A cells at indicated concentrations. Transduction of BoNT/B into N2A cells using Lipofectamine LF3000 (LF3000) allows VAMP2 cleavage. Note the absence of VAMP fragments at all BoNT/B concentrations. (C) A polyclonal antibody against synaptotagmin 1 reveals its presence in the SiMa and N2A cell lines. (D) The SiMa but not N2A cell line expresses neuronal ganglioside GT1b as revealed by specific anti-GT1b antibody.

FIGURE 2

Generation of GFP-VAMP2 SiMa cell line using viral transduction. (A) Schematic showing the fusion protein containing GFP inserted at the amino terminus of VAMP2. TMR, transmembrane region. (B) GFP-VAMP2 localizes to vesicular structures (top) and is released into the cytosol (bottom) upon incubation with BoNT/B (30 nM). (C) Robust expression following viral transduction and puromycin selection is evident in GFP and differential interference contrast (DIC) merged images. (D) Stable GFP-VAMP2 cleavage product can be detected following incubation with BoNT/B (30 nM). Immunoblotting was performed using a GFP antibody.

FIGURE 3

Generation of stable SiMa cell lines with VAMP2 fusions to peroxidase APEX2 and luciferase NanoLuc. (A) Immunoblot showing expression of APEX2- and NanoLuc-VAMP2 constructs in SiMa cells with expected molecular weights of ∼45 kD and ∼30 kD. Both constructs carry the HA tag as evidenced by immunoblotting. (B) The fusion proteins exhibit robust expression as revealed by immunocytochemical analysis using antibodies against the HA-tag. (C) Immunoblot demonstrating that the APEX2-VAMP2 reporter molecule is resistant to cleavage by BoNT/B (10 nM), whereas VAMP cleavage is detectable in the differentiated NanoLuc-VAMP2 cells. The arrow indicates the cleaved product.

FIGURE 4

Detection of BoNT/B activity in NanoLuc-VAMP2 SiMa cells using specific BoNT/B-cleaved VAMP2 antibody. (A) The VAMP2 amino acid sequence used for generation of the rabbit polyclonal antibody is underlined. The arrow indicates the cleavage site by BoNT/B. (B) Immunoblot showing dose-dependent increase of BoNT/B-cleaved NanoLuc-VAMP2 product using the cleaved VAMP2 antibody. Engineered SiMa cells were incubated for 60 h in the presence of indicated concentrations of BoNT/B followed by SDS–PAGE and immunoblotting. (C) Graph showing quantification of dose-dependent increase in cleaved VAMP2 immunoblotting signal in response to BoNT/B with sensitivity in the low picomolar range (n = 3 ± SD).

FIGURE 5

One-step ELISA assay using the NanoLuc-VAMP2 construct. (A) Schematic (left) showing the principle of one-step ELISA. The cleaved VAMP2 antibody was immobilized on Protein A plates for capture of the BoNT/B-cleaved VAMP2 construct. (B) Graph showing the dose-dependent detection of NanoLuc-VAMP2 cleavage by Metabiologics BoNT/B (n = 3 ± SD). (C) The NanoLuc assay allows detection of the VAMP cleavage by BoNT/B at 0.1 mouse LD₅₀ above the baseline (n = 3 ± SD, ^∗∗p < 0.01, Student’s t-test).