Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H

Abstract

Background. Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.Methods. We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay.Results. Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10−8 M) than its BoNT/F affinity (KD = 9.1 × 10−11 M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD = 1.48 × 10−10 M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody.Conclusions. This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

Impact of Clostridium botulinum genomic diversity on food safety

“…Hybrids of type C and type D neurotoxin (type C/D or type D/C) are formed more frequently than type C or type D neurotoxin [30]. A highly novel neurotoxin (type FA (Figure 1), also known as type H and type HA) that contains regions of similarity to type A neurotoxin and type F neurotoxin has been recently identified and is being characterised [31, 32, 33, 34, 35, 36]. Hybrid neurotoxin subtypes have been identified, e.g.…”

Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

“…Subsequently, it was confirmed that this new toxin cleaves VAMP-2 (also called synaptobrevin 2) between L54 and E55 [21,24], which is identical to the behavior of BoNT/F5, but different from all other BoNT/F subtypes that cut VAMP-2 between Q58 and K59 [25]. Interestingly, only one of the six anti-BoNT/F antibodies tested in a recent study showed binding to the new toxin, albeit weak (K D ~75 nM), suggesting that it is immunologically different from BoNT/F [22]. In contrast, monoclonal antibodies RAZ1 and CR2 (both target the H C of BoNT/A) precipitated the new toxin from the culture supernatant [24] and neutralized the new toxin in a mouse bioassay [22].…”

“…Furthermore, several polyclonal antibodies raised against BoNT/A1 were found to neutralize this toxin, but at a lower potency compared to BoNT/A1 [21]. A new potent monoclonal antibody directed against this new toxin was reported in 2016 [22]. …”

“…Interestingly, only one of the six anti-BoNT/F antibodies tested in a recent study showed binding to the new toxin, albeit weak (K D ~75 nM), suggesting that it is immunologically different from BoNT/F [22]. In contrast, monoclonal antibodies RAZ1 and CR2 (both target the H C of BoNT/A) precipitated the new toxin from the culture supernatant [24] and neutralized the new toxin in a mouse bioassay [22]. Based on these data, we suggested to name this new toxin as BoNT/HA [26].…”

“…In our previous study, we showed that the H C of BoNT/HA (H C HA) binds weakly to the protein moiety of its cell-surface receptor SV2C when compared to H C A1 in spite of high sequence similarity [26]. Furthermore, a highly potent anti-BoNT/A antibody CR2, which is currently in clinical trials, displayed a ~540-fold decreased affinity on BoNT/HA according to a recent neutralization study [22]. These findings thus raised questions on how BoNT/HA behaves differently than BoNT/A1 on neuronal receptor binding and responds differently to the known antitoxins.…”