Russell G.A. Jones

Detection of antibodies against botulinum toxins

After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact, the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin‐specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD50 bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of <0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype‐specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells.

Measurement of botulinum types A, B and E neurotoxicity using the phrenic nerve-hemidiaphragm: Improved precision with in-bred mice

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. The current method for assessing the potency of botulinum toxin and antitoxins is the mouse LD50 assay. The mouse phrenic nerve-diaphragm assay is an in vitro assay that closely mimics in vivo respiratory paralysis. In this study, we have further improved the assay by using gelatin as a non-frothing alternative to albumin and investigated the effects of botulinum toxin serotypes A, B and E on phrenic nerve-hemidiaphragms from out-bred MF1 and in-bred Balb/c mice. Improved reproducibility was found with in-bred mice. Balb/c mice were also found to be much less sensitive to type B toxin perhaps indicating differences in the expression of receptor components. Hemidiaphragm preparations from Balb/c mice were approximately 7 times more sensitive to type A toxin and 7-12 times more sensitive to type E toxin relative to type B toxin. These findings indicate that when fully optimised the mouse nerve-diaphragm preparation can provide a functional in vitro model for accurate and reproducible assessment of toxin activity.

An improved method for development of toxoid vaccines and antitoxins

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylation with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600-times higher than those produced with formaldehyde toxoid, despite generating equivalent ELISA antitoxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxins structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins and immunogens.

Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies.

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4-7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.

Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin

BackgroundBotulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use.ResultsDiversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8.ConclusionsThe presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate antibody fragments with therapeutic potential.

New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, C1 and E intra-cellularly cleave SNAP25 and/or Syntaxin (type C1 only) resulting in a flaccid paralysis. Although highly sensitive, robust in vitro endopeptidase immunoassays have been developed for some serotypes, an endopeptidase immunoassay for type C1 has not previously been described. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and a new specific antibody to the SNAP25(191-198) octapeptide epitope that becomes exposed following cleavage by type C1 toxin. The highly specific nature of the detecting antibody was illustrated by the failure of anti-SNAP25(191-198) to recognise the type A cleavage product which differs by just one amino acid residue. Conversely, anti-SNAP25(190-197), which recognises the type A cleavage product, fails to cross react with the type C1 toxin cleavage product. Utilising Syntaxin(232-266) peptide substrate, and a specific antibody to the cleavage product epitope, Syntaxin(254-261), it was also possible to develop an endopeptidase immunoassay. Assay sensitivities allowed the detection of less than 0.1 LD(50)/ml (25 pg/ml) of type C1 haemagglutinin-complexed toxin. The assay failed to detect toxin serotypes A, B, D, E, F or G and therefore also provides an alternative highly specific in vitro identity test. In the absence of trypsin inhibitors, the assay is also capable of detecting 2 pg/ml of trypsin activity, or trypsin like contaminants. These new immunoassays will therefore provide highly specific tools for monitoring botulinum toxin light chain endopeptidase activity and serotype identity.

Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: Detection with a new neoepitope endopeptidase immunoassay

Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.

Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype B

ABSTRACT New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 107 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.

Use of a new functional dual coating (FDC) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the carboxy-terminal end consisting of a receptor-binding (HC) domain, while the amino-terminal (HN) domain is linked by a critical disulfide bond to a light chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60 % of botulism patients. A novel FDC (functional dual coating) microtitre plate immuno-biochemical assay, which quantifies botulinum toxicity by measuring the HC domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD50 per ml serum or 0.13 pg ml(-1) using just 100 µl of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD50 per ml serum of type A toxin were quantified for the first time using the modified FDC assay in two severely intoxicated UK patients who required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBA-negative sample was found to contain 0.32 LD50 per ml extract. The FDC assay provides a real alternative for public health laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.