Transcutaneous immunisation assisted by low-frequency ultrasound.

Low-frequency ultrasound application is known to increase the skin's 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

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of “superiority” of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

Purpose. In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. Methods. TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. Results. TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DTalum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. Conclusions. The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants. KEY WORDS: cholera toxin; CpG; diphtheria toxoid; microneedle array; transcutaneous immunization

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

Rational arguments for regulatory acceptance of consistency testing benefits of non animal testing over in vivo release testing of vaccines

INTRODUCTION: There are rational arguments to replace existing in vivo potency and safety assays for batch release testing of vaccines with more advanced non-animal techniques to measure critical quality attributes. However, introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is&nbsp;challenging. AREAS COVERED: This report describes the hurdles encountered in substituting assays and ways to overcome these and provides arguments why more advanced alternatives are superior, not only as tool to monitor the quality of vaccines but also from a practical, economical and ethical point of view. The rational arguments provided for regulatory acceptance can support a strategy to replace/substitute any batch release test if an appropriate non-animal testing strategy is&nbsp;available. EXPERT OPINION: For several vaccines, in vivo release assays have been replaced leading to an optimized control strategy. For other vaccines, new assays are being developed, that can expected to be introduced within 5-10 years. From a scientific, logistical and animal welfare perspective, it would be beneficial to substitute all existing in vivo batch release assays for vaccines. Given the challenges relating to development, validation and acceptance of new methods, and considering the relatively low prices of some legacy vaccines, this cannot be done without government incentives and supportive regulatory authorities from all&nbsp;regions.</p

Investigating Alternative Container Formats for Lyophilization of Biological Materials Using Diphtheria Antitoxin Monoclonal Antibody as a Model Molecule

When preparing biological reference materials, the stability of the lyophilized product is critical for long-term storage, particularly in order to meet WHO International Standards, which are not assigned expiry dates but are expected to be in use for several decades. Glass ampoules are typically used by the National Institute for Biological Standards and Control (NIBSC) for the lyophilization of biological materials. More recently, a clear need has arisen for the filling of smaller volumes, for which ampoules may not be optimal. We investigated the use of plastic microtubes as an alternative container for small volume fills. In this study, a recombinant diphtheria antitoxin monoclonal antibody (DATMAB) was used as a model molecule to investigate the suitability of plastic microtubes for filling small volumes. The stability and quality of the dried material was assessed after an accelerated degradation study using a toxin neutralization test and size exclusion HPLC. While microtubes have shown some promise in the past for use in the lyophilization of some biological materials, issues with stability may arise when more labile materials are freeze-dried. We demonstrate here that the microtube format is unsuitable for ensuring the stability of this monoclonal antibody