Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin

Abstract

Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A-G) which differ from each other by 34-64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.

Fig. 1

Amino acid sequence alignment of cross-serotype binding antibodies. The V_H and V_k genes of cross-serotype reactive scFv were aligned. CDR, complementarity determining region; FR, framework region. CDR and FR defined according to IGMT (http://imgt.cines.fr/IMGT_vquest/share/textes).

Fig. 2

Strategies used to increase affinity and broaden specificity of 1B18 and 4E17 antibodies. The method used to introduce diversity is indicated (L_C shuffling using the Fab or scFv format or by creating libraries of VHCDR1, VHCDR2 or VHCDR3 mutants). The BoNT serotype used for selection is also indicated. Where more than one serotype is indicated, selections were done sequentially on the serotypes indicated. The selection outcome is also indicated: K_D < BoNT/X indicates that higher affinity antibodies were generated to the BoNT serotypes indicated. No mAbs isolated indicates that no mAbs binding BoNT/C were isolated. Failed indicates that no antibodies were generated that had higher affinities for BoNT/B and E than 4E17.4.

Fig. 3

1B18 and 4E17 bind overlapping epitopes. Yeast-displayed 1B18 scFv was incubated with either BoNT/A (top two panels) or BoNT/B (bottom two panels) followed by incubation with either 4E17.1 IgG or the BoNT/A antibody RAZ1 or the BoNT/B antibody B11E8. 4E17.1 and 1B18 cannot simultaneously bind BoNT/A or B.

Fig. 4

Mapping the binding site of 1B18 and 4E17.1 mAbs by using yeast-displayed BoNT domains. The H_C, H_N and L_C domains of BoNT/A, B and E were displayed on the surface of yeast and stained with 1B18 or 4E17.1 IgG. The level of BoNT domain display was quantitated using a mAb to a SV5 epitope tag fused to the C-terminus of the domain. Both mAbs bound only to the H_N (1B18 to BoNT/A and B H_N and 4E17.1 to BoNT/A, B and E H_N).

Fig. 5

Fine epitope of 1B18 and 4E17.1 mAbs. Binding of 1B18 and 4E17.1 mAbs to wild-type BoNT/B H_N and the BoNT/B H_N E747A mutant. As a control binding of the H_N mAb 1B11 is also shown. 1B11 binds to both the wild-type and mutant H_N, whereas neither 1B18 nor 4E17.1 bind to the H_N E747A mutant. The results indicate that the fine epitope of both 1B18 and 4E17.1 is located around H_N amino acid E747.

Fig. 6

Model of the functional binding epitopes of 1B18 and 4E17.1 mAbs. (A) The epitopes of 1B18 on BoNT/B (center panels) and 4E17.1 on BoNT/A (left panels) and BoNT/E (right panels) are indicated. The X-ray crystal structures of BoNT/A (green), BoNT/B (cyan) and BoNT/E (magenta) were structurally aligned on amino acid 757 of BoNT/A and are shown as ribbon diagrams. The middle set of panels represent the upper panels rotated 90° to give a birds-eye view of the mAb epitope. The 1B18 and 4E17 epitopes are shown in surface projection colored from red to wheat to indicate the ΔΔG values from Table IV. The lower set of panels show the side chains of amino acids E755, E756 and E757 of BoNT/A and their corresponding amino acids in BoNT/B and E on the aligned crystal structures.

Fig. 7

Antibody protection of mice against challenge with BoNT/B or E. Twenty-five micrograms of the indicated mAb was mixed with 200 mouse LD₅₀ of either BoNT/B (4B6.1 and 2B18.1) or BoNT/E (4E17.1) and injected intraperitoneally into each of 10 mice. The number of mice surviving versus time is plotted. The toxin only control is for BoNT/B.