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.

Dual Specificity of Anti-CXCL10-CXCL9 Antibodies Is Governed by Structural Mimicry

“…As such, a first set of rabbit CXCL10 sitedirected mutants was generated as follows: rab10S 13 Q 17 , rab10F 35 P 37 , rab10K 48 , and rab10S 58 N 63 V 68 KRSP 74 -77 also termed rab10Cterm. Similarly, three cynomolgus CXCL9 sitedirected mutants were designed as follows: cyn9S 13 , cyn9S 33 P 34 , and cyn9R 98 T 103 . Mutated proteins were expressed as soluble NusA fusion proteins, using a pET43-derived expression vector, and purified by affinity chromatography via their histidine tag, as described elsewhere ( Fig.…”

“…Site-directed Mutagenesis-Five rabbit CXCL10 mutants, rab10S 13 , rab10K 48 , rab10S 58 N 63 V 68 KRSP 74 -77 , rab10Q 17 , and rab10S 13 , and three cynomolgus CXCL9 mutants, cyn9S 13 , cyn9S 33 P 34 , and cyn9R 98 T 103 , were generated by site-directed mutagenesis. Residues were numbered according to the target sequence.…”

Cluster of two cases of botulism due to Clostridium baratii type F in France, November 2014

“…The existence of considerable sequence variability within BoNT serotypes can significantly affect antibody binding and neutralisation [20,24]. There are reports of various levels of cross neutralisations of BoNT F with antitoxin E in animal models [3,4,25,26].…”

“…Two mAbs were identified capable of binding with high affinity different BoNTs in vitro and capable of neutralising BoNT B and E in vivo [24].…”

Phage and Yeast Display

“…In addition to novel mAb discovery, yeast surface display approaches are used for affinity maturation of known anti-BoNT antibodies. Yeast display V L shuffled libraries of two previously identified scFvs were sorted on decreasing concentrations of BoNT/B, E, or F and produced improved mAbs with broadened high affinity binding to additional subtypes beyond the parent clone (116). Similarly, V L shuffling through haploid yeast mating methods generated affinity-matured Fabs from the parent scFvs isolated from an immune yeast library (117).…”