A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

Abstract

Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA

“…Since the BoNT/H LC-H N is most homologous to the BoNT/F5 subtype, we sorted five yeast-displayed scFv libraries generated from mice immunized with recombinant BoNT/F domains (Table 1) [18] to determine if rare cross-reactive BoNT/H LC-H N mAbs could be isolated. After sorting on BoNT/H LC-H N , a total of six unique scFv were isolated with K D values of 20.5 nM-1490 nM (median of 127 nM, Table 2).…”

“…The BoNT/H fragments BoNT/H LC (1-444) and HLC-H N (1-859) and soluble scFv were expressed and purified as described [18]. For ELISA, 100 µL of serially diluted BoNT/H LC-H N was coated on a plate, with F1 LC-H N as a control, followed by incubation with mAb 6F5.4 or anti-His tag IgG (1 µg/mL) with detection by using HRP-labeled goat anti-human or mouse IgG (0.1 µg/mL, Jackson ImmunoResearch) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate.…”

“…Individual colonies were picked from SD-CAA plates, cultured and scFv display induced for further characterization [31]. Libraries made from mice immunized with BoNT/F fragments [18] were sorted as described above (Table 1).…”

“…Flow cytometry was used because it allows measurement of the scFv dissociation equilibrium constant (K D ) without the need to subclone, express and purify the scFv as would be required for ELISA or surface plasmon resonance. The dissociation equilibrium constant (K D ) of scFv was determined by using flow cytometer as described [18].…”

“…Epitope groups on BoNT/H surface were deduced from competition assays as described [18]. Briefly, yeast-displayed scFvs were incubated with 100 nM of BoNT/H LC-H N followed by incubation with soluble myc-tagged scFv.…”