Paul Stickings

A review of the international issues surrounding the availability and demand for diphtheria antitoxin for therapeutic use

Diphtheria treatment requires early administration of diphtheria antitoxin (DAT), an immunoglobulin preparation that neutralises circulating diphtheria toxin. Here, we review issues relating to the supply and use of DAT and assess its availability by means of an international survey. Results showed that several countries do not currently hold DAT stockpiles due to low prevalence, and hence perceived risk of diphtheria, and/or difficulties in obtaining DAT supplies. The potential for importation of cases into any country exists globally, since diphtheria remains endemic in many regions. It is therefore important that DAT be readily available - particularly since waning diphtheria immunity has been observed among adult populations in countries with good vaccination coverage. Options for diphtheria therapy are discussed.

Transcutaneous immunisation assisted by low-frequency ultrasound

Low-frequency ultrasound application is known to increase the skins permeability to large molecules such as vaccines, and to enable transcutaneous immunisation. Sodium dodecyl sulphate (SDS) - a skin irritant - is often included in the coupling medium at 1% (w/v), as this has been found to enhance skin permeability. In this paper we show, for the first time, the feasibility of low-frequency ultrasound-assisted transcutaneous immunisation in the absence of SDS. Antibody titres were strongly influenced by experimental conditions. SDS presence in the coupling medium increased antibody titres, though a lower concentration of 0.5% (w/v) generated much higher titres than the commonly used 1% (w/v), despite causing less skin damage. A lower ultrasound duty cycle of 10% generated higher antibody titres than a duty cycle of 20%, also despite causing lower skin damage. Such lack of correlation between skin damage and immune responses indicates that enhancement of skin permeability to topically applied antigen (as indicated by changes in skin integrity) was not the main mechanism of low-frequency ultrasound-assisted skin immunisation.

Development and use of a novel in vitro assay for testing of diphtheria toxoid in combination vaccines.

Testing of diphtheria vaccines for routine lot release relies heavily on the use of in vivo potency assays. However, consistency of production is also recognised as an important feature of vaccine manufacture, and in vitro assays are superior to in vivo assays for providing this information. In adsorbed vaccines, antigen and adjuvant are the major components contributing to immunogenicity and are therefore critical factors to be evaluated as part of consistency testing. Here we describe a simple and sensitive Enzyme Linked Immunosorbent Assay (ELISA) which has been developed to quantify diphtheria toxoid antigen in combined vaccine products and can also be used to monitor the degree of adsorption. This assay can be applied to a variety of multi-component vaccines and is robust, specific and highly sensitive, with a limit of quantification of approximately 0.005 Lf/ml. The antigen assay is an excellent test to characterise vaccines and monitor trends in production. For well established vaccines, the antigen assay could be used alongside other in vitro methods to provide a more informative product profile, with the ultimate aim of reducing the requirement for in vivo potency assays and therefore the number of animals required for routine batch release testing.

External quality assessment for the determination of diphtheria antitoxin in human serum.

ABSTRACT Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro “gold standard.” The performance of laboratories using NT was generally very good, while the laboratories’ performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

Transcutaneous Immunization with Cross-Reacting Material CRM197 of Diphtheria Toxin Boosts Functional Antibody Levels in Mice Primed Parenterally with Adsorbed Diphtheria Toxoid Vaccine

ABSTRACT Transcutaneous immunization (TCI) capitalizes on the accessibility and immunocompetence of the skin, elicits protective immunity, simplifies vaccine delivery, and may be particularly advantageous when frequent boosting is required. In this study we examined the potential of TCI to boost preexisting immune responses to diphtheria in mice. The cross-reacting material (CRM197) of diphtheria toxin was used as the boosting antigen and was administered alone or together with either one of two commonly used mucosal adjuvants, cholera toxin (CT) and a partially detoxified mutant of heat-labile enterotoxin of Escherichia coli (LTR72). We report that TCI with CRM197 significantly boosted preexisting immune responses elicited after parenteral priming with aluminum hydroxide-adsorbed diphtheria toxoid (DTxd) vaccine. In the presence of LTR72 as an adjuvant, toxin-neutralizing antibody titers were significantly higher than those elicited by CRM197 alone and were comparable to the functional antibody levels induced after parenteral booster immunization with the adsorbed DTxd vaccine. Time course study showed that high levels of toxin-neutralizing antibodies persisted for at least 14 weeks after the transcutaneous boost. In addition, TCI resulted in a vigorous antigen-specific proliferative response in all groups of mice boosted with the CRM197 protein. These findings highlight the promising prospect of using booster administrations of CRM197 via the transcutaneous route to establish good herd immunity against diphtheria.

In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009. The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.

A Cell Line for Detection of Botulinum Neurotoxin Type B

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products.