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.

Calibration of the Ph. Eur. Biological Reference Preparation (BRP) for tetanus vaccine (adsorbed) batch 3.

A joint collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC) to establish replacement batches for the European Pharmacopoeia (Ph. Eur.) Tetanus Vaccine (adsorbed) Biological Reference Preparation (BRP) batch 2 and for the WHO 3rd International Standard (IS) for Tetanus toxoid (adsorbed). Two freeze-dried stabilised tetanus vaccine (adsorbed) candidate preparations (Preparation A, 08/218 and Preparation B, 08/102) were calibrated against the current 3rd IS/BRP batch 2 (Preparation C) using challenge methods in guinea pigs and mice as described in the Ph. Eur. general chapter 2.7.8. Assay of tetanus vaccine (adsorbed). They were also assayed by serology methods. The WHO 2nd IS for Tetanus toxoid adsorbed (TEXA-2) was additionally included in the sample panel as Preparation D. Thirty-four laboratories (regulatory organisations and manufacturers) from 22 countries participated in the collaborative study. The majority of participants performed 2 independent challenge tests. Nine laboratories performed challenge assays in guinea pigs and 30 laboratories performed challenge assays in mice. Eight laboratories performed serology in guinea pigs and 1 laboratory performed serology in mice. For Preparation A, the geometric mean (GM) potency estimate (with 95 % confidence interval (CI)) in guinea pigs for all laboratories that provided valid results (n = 6) was 488.5 (354.2-673.6) IU/ampoule. For valid mouse assays (n = 25) the GM potency (with 95 % CI) was 259.8 (223.5-302.0) IU/ampoule. The inter-laboratory geometric coefficient of variation (GCV) was 36 % for guinea pig assays and 45 % for mouse assays. This compared favourably with the calibration of the 3rd IS/BRP batch 2 where the inter-laboratory GCV was 36 % and 42 % in guinea pigs and mice, respectively. For Preparation B, the GM potency estimate (with 95 % CI) in guinea pigs for all laboratories that provided valid results (n = 6) was 107.9 (64.1-181.7) IU/ampoule. For valid mouse assays (n = 24) the GM potency (with 95 % CI) was 147.9 (126.3-173.1) IU/ampoule. The inter-laboratory GCV was 64.3 % for guinea pig assays and 45.2 % for mouse assays. From the collaborative study, Preparation A appeared more suitable to be the replacement Ph. Eur. BRP as it is similar to the Tetanus vaccine (adsorbed) BRP batch 2, except for nature of the stabiliser. Preparation A was confirmed to have higher potency, readily detectable tetanus toxoid, and confirmed satisfactory stability and performance in challenge assays. Preparation A was adopted in January 2011 by the Ph. Eur. Commission as the Tetanus vaccine (adsorbed) BRP batch 3, with assigned potencies of 490 IU/ampoule in the guinea pig challenge assay and of 260 IU/ampoule in the mouse challenge assay. The same Preparation A was adopted in October 2010 as the WHO 4th IS for Tetanus toxoid (adsorbed), with the assigned activity of 490 IU/ampoule from guinea pig challenge assays. A follow-up study (reporting study) was organised by the EDQM to assess the impact of the potency assigned to the BRP batch 3 for mouse challenge assays on the outcome of batch release testing in Europe. Eight laboratories including official medicines control laboratories (OMCLs) and manufacturers reported the results of their routine testing, using the BRP batch 3 in addition to their regular reference preparation. For each tested product, participants calculated the potency relative to their routine reference and relative to the BRP batch 3. No common sample panel was distributed to participants. In total, data on 40 batches of different marketed tetanus vaccines were reported. Overall, a good concordance was observed between the potencies calculated relative to the BRP batch 2 and relative to the BRP batch 3. On average, the potency estimates were 10 % lower when expressed relative to the BRP batch 3. Cases of discrepant decisions for batch release were very limited and affected mainly batches with specifications close to the pharmacopoeial requirements. The reasons for differences in estimated potencies are discussed. The study showed that the use of the BRP batch 3 with an assigned potency of 260 IU/ampoule does not result in substantial change in the potency of different marketed products. This confirmed that the mouse challenge potency value assigned to the BRP batch 3 is suitable.

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.

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.

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.

Optimization of the formulation for a candidate lyophilised tetanus toxoid reference preparation.

Tetanus toxoid is a vital primary reference material used for standardization of assays required to establish the antigenic purity of tetanus toxoid for vaccine production. Several formulations were assessed and ampouled fills of each formulation lyophilised. The relative Lf content determined by Ramon flocculation, SRD, and ELISA assays was measured. The stability of the tetanus toxoid activity in each formulation was assessed by accelerated degradation studies. Formulations containing glycine were not suitable in flocculation tests but both sorbitol and trehalose formulations were. The trehalose/sodium chloride formulation had a good appearance, showed good activity in all assays and maintained its activity best under stress conditions. This formulation has been applied to a large scale batch of ampoules prepared as a WHO candidate replacement standard, evaluated in a collaborative study and accepted as a replacement WHO IS for use in flocculation test (WHO ECBS, October 2007, ref no BS/07.2061). The stability of this formulation was also excellent for the large scale batch. The benefits of using thermal analysis and freeze drying microscopy coupled with small scale lyophilisation trials in order to screen formulations for the preparation of batches of biological reference materials are demonstrated.

Calibration of European pharmacopoeia biological reference preparation for diphtheria vaccine (adsorbed) batch 4.

A collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the National Institute for Biological Standards and Control (NIBSC) to establish replacement batches of the current World Health Organization (WHO) International Standard (IS) and European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Diphtheria Vaccine (Adsorbed). Two candidates were assayed against the current 3rd IS/BRP batch 3 for Diphtheria Vaccine (Adsorbed) with an assigned potency of 160 IU/ampoule using established WHO/Ph. Eur. challenge methods in guinea pigs as described in the Ph. Eur. general chapter 2.7.6. Assay of diphtheria vaccine (adsorbed). Twenty-one laboratories (regulatory organisations and manufacturers) from 17 countries participated in the study. Two freeze-dried, stabilised diphtheria vaccine (adsorbed) preparations were included in the study: Preparation A (07/218) and Preparation B (07/216). As stocks of the 3rd IS were very low, the Diphtheria vaccine (adsorbed) BRP batch 3, which is identical to the 3rd IS but which was kept at the EDQM, was used for the calibration (coded Preparation C). The majority of participants performed 2 independent challenge tests. Five laboratories performed the intradermal challenge test, 16 laboratories performed the systemic challenge test. For Preparation A, the unweighted geometric mean potency estimate (with 95 % confidence limits) for all laboratories that provided valid results (n = 17) was 97.2 (89.5-105.6) IU/ampoule. For systemic challenge assays (n = 14) the unweighted geometric mean potency was 97.0 (88.1-106.7) IU/ampoule. The between-laboratory GCV was 17.4 % for all assays and 18.0 % for systemic challenge assays. There was no significant difference in estimates for intradermal or systemic challenge (p = 0.45). For Preparation B the unweighted geometric mean potency estimate (with 95 % confidence limits) for all laboratories that provided valid results (n = 19) was 213.4 (185.7-245.4) IU/ampoule. For systemic challenge assays (n = 14) the unweighted geometric mean potency was 202.0 (170.4-239.5) IU/ampoule. The between-laboratory GCV was 33.5 % for all assays and 34.3 % for systemic challenge assays. For both preparations there is a good agreement between results obtained from systemic and intradermal challenge methods. Greater between-laboratory variability was observed for systemic assays than for intradermal challenge assays, although the small number of intradermal assays performed in the study makes comparison difficult. The GCV for all assays was 17.4 % for potency estimates of Preparation A and 33.5 % for Preparation B. This compares favourably with the calibration of the current standard where the between-laboratory GCV for all assays was 28.3 %. From the collaborative study both Preparation A and Preparation B appeared suitable to replace the current Diphtheria vaccine (adsorbed) BRP batch 3. Due to the similarity of Preparation A with the current BRP batch 3, the Ph. Eur. Commission adopted Preparation A as Diphtheria vaccine (adsorbed) BRP batch 4 with an assigned potency of 97 IU/ampoule.

Collaborative study on a Guinea pig serological method for the assay of acellular pertussis vaccines.

An international collaborative study (coded BSP083) was performed under the aegis of the Biological Standardisation Programme supported by the Council of Europe and the European Commission, with the aim of replacing the in vivo challenge assays for potency determination of combined acellular pertussis (aP) vaccines by a refined procedure also allowing reduction of animal use. This study investigates whether the immunogenicity of aP vaccine components could be assayed in a guinea pig (gp) serology model, using the same vaccine immunising doses as for D and T components potency testing, instead of using separate animals as is currently done. The BSP83 project is a follow up of 3 former collaborative studies (coded BSP019, BSP034 and BSP035) on serological methods for the potency testing of tetanus (T) and diphtheria (D) vaccines for human use. The use of gp instead of mice serology has the advantage of providing a larger volume of good quality antiserum for the assay of several vaccine components in the same sample, hence providing the opportunity for animal sparing. The results of Phase I of the study demonstrated that gp serology may be a useful method for the immunogenicity assay of acellular pertussis vaccines. This was confirmed in Phase II of the study, using 7 different combined aP vaccines in an international collaborative study involving 17 laboratories from both public and private sectors. Clear dose-response relationships were observed for different vaccines by ELISA, for antibodies against aP antigens, i.e. pertussis toxin (PT), filamentous haemagglutinin (FHA), fimbrial agglutinogens-2/3 (Fim 2/3) and pertactin (PRN). Intra- and inter-laboratory variations of aP ELISA results were found to be within an acceptable range. For some combined vaccines, however, the range of vaccine dilutions for immunisation confirmed to be optimal for D and T potency testing may not provide optimal dose-response for all aP components. Method adjustments may thus be required and suitability should therefore be demonstrated for each vaccine combination and product prior to the application of this assay. The results of this study support the use of the gp serological method for the determination of the immunogenicity of aP vaccines. The application of the method for batch release testing of combined D, T and aP vaccines could significantly contribute to the implementation of the 3R principles through reduction of animal use and improved animal welfare, whilst reducing costs. As an outcome of this study, the Group of Experts No. 15 on Sera and Vaccines of the European Pharmacopoeia (Ph. Eur.) decided in February 2009 to include the gp serological assay as an example in the Ph. Eur. General chapter 2.7.16. on acellular pertussis vaccine assay.

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test.

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.

Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan (TMC) nanoparticles for non-invasive vaccine delivery.

Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan is a cationic biopolymer which exerts advantages as a vaccine carrier due to its immune stimulating activity and bioadhesive properties that enhance cellular uptake and permeation as well as antigen protection. Similar effects are also shown by chitosan derivatives. In this study, the nanoparticulate systems were prepared by using differently charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic), and mono-N-carboxymethyl chitosan (MCC, polyampholytic) for mucosal immunisation. The derivatives were synthesised and characterised in-house. The aqueous dispersions of the derivatives were also prepared for comparison. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, chitosan and TMC. Nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). Nanoparticles with high loading efficacy (>90% m/m), particle size within the range of 40-400nm, with a negative surface charge for MCC and positive surface charge for TMC and chitosan were obtained. The structural integrity of the TT in the formulations was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune responses induced by the formulations loaded with tetanus toxoid were studied in vivo in Balb/c mice. Enhanced immune responses were obtained with intranasal (i.n.) application of nanoparticle formulations. Chitosan and TMC nanoparticles which have positively charged surfaces induced higher serum IgG titres when compared to those prepared with MCC which are negatively charged and smaller in size. Nanoparticle formulations developed in this study can be used as promising adjuvant/delivery systems for mucosal immunisation.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

Botulinum type A toxin neutralisation by specific IgG and its fragments: a comparison of mouse systemic toxicity and local flaccid paralysis assays.

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin neutralising activity of specific immunoglobulin G (IgG) and its fragments (F(ab')(2), Fab', Fab) purified from pentavalent botulinum antisera raised in goats. Each assay method was repeated on three separate occasions in mice and relative potencies calculated with respect to a type A equine reference antitoxin. The conventional assay, which measures the number of mice surviving typically after 72 or 96 h following the intraperitoneal administration of a mixture of toxin and antitoxin, gave the following order of potency IgG>F(ab')(2)>Fab'>Fab (6.8>4.7>3.5>2.6 IU/mg). Differences in potency are likely to be due to differences in the pharmacokinetics of the antitoxins, which are related to their molecular weight. The alternative local flaccid paralysis assay, where toxin and antitoxin are injected subcutaneously into the left inguinocrural region, gave results with a narrower range of activities: IgG>Fab'>F(ab')(2)>Fab (6.0>5.9>5.5>4.6 IU/mg). Comparison of the two assay methods showed no significant differences for IgG, F(ab')(2) or Fab', although the Fab fragment was significantly more potent in the non-lethal assay probably because of the reduced influence of antitoxin pharmacokinetics in this localised assay. These findings show that a local flaccid paralysis assay provides a less time consuming and more humane alternative to the lethal assay for the potency testing of botulinum IgG and F(ab')(2) antitoxins.

Regulatory considerations on new adjuvants and delivery systems.

New and improved vaccines and delivery systems are increasingly being developed for prevention, treatment and diagnosis of human diseases. Prior to their use in humans, all new biological products must undergo pre-clinical evaluation. These pre-clinical studies are important not only to establish the biological properties of the material and to evaluate its possible risk to the public, but also to plan protocols for subsequent clinical trials from which safety and efficacy can be evaluated. For vaccines, evaluation in pre-clinical studies is particularly important as information gained may also contribute to identifying the optimum composition and formulation process and provide an opportunity to develop suitable indicator tests for quality control. Data from pre-clinical and laboratory evaluation studies, which continue during clinical studies, is used to support an application for marketing authorisation. Addition of a new adjuvant and exploration of new delivery systems for vaccines presents challenges to both manufacturers and regulatory authorities. Because no adjuvant is licensed as a medicinal product in its own right, but only as a component of a particular vaccine, pre-clinical and appropriate toxicology studies need to be designed on a case-by-case basis to evaluate the safety profile of the adjuvant and adjuvant/vaccine combination. Current regulatory requirements for the pharmaceutical and pre-clinical safety assessment of vaccines are insufficient and initiatives are in place to develop more specific guidelines for evaluation of adjuvants in vaccines.

A review of WHO International Standards for botulinum antitoxins.

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (A-G). These toxins can cause a life threatening systemic toxicity whether through natural causes such as food poisoning, infant botulism, wound botulism, or through use as bio-terror agents (e.g. inhalational botulism). It was realised early on that standard reference botulinum antitoxins were required to reduce the variation between assays and ensure a consistent potency of therapeutic antitoxins and vaccines, and to define the serotype. This led to the International Unit being defined by the World Health Organisation (WHO) in the 1960s with the establishment of the first International Standards (IS) for serotypes A-F. Since then botulinum antitoxin ISs have been used world wide as the 'yard stick' to measure the neutralising potency of antitoxins. These primary WHO ISs are used to calibrate in house working reagents that are more extensively utilised. A definition of the International Unit for serotype G antitoxin has yet to be defined or accepted by the WHO and urgently needs addressing. However, before September 11th 2001 there was very little interest in botulinum antitoxin IS and as a result stocks of most of the original preparations are now completely exhausted or depleted and replacements long overdue. We have reviewed the extensive history and availability of the primary WHO ISs and interim materials. All type A and B antitoxin materials were recently assayed and their relative activities confirmed against the original IS preparations. The recent increase in demand for these materials has further exacerbated the shortage. We describe here the production and characterization of stable freeze dried potential candidate replacements along with a new prospective first IS for type G antitoxin. Available toxin A reference preparations are also briefly reviewed.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccine (part 2).

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

Effect of skin barrier disruption on immune responses to topically applied cross-reacting material, CRM(197), of diphtheria toxin.

The high accessibility of the skin and the presence of immunocompetent cells in the epidermis makes this surface an attractive route for needle-free administration of vaccines. However, the lining of the skin by the stratum corneum is a major obstacle to vaccine delivery. In this study we examined the effect of skin barrier disruption on the immune responses to the cross-reacting material CRM(197), a nontoxic mutant of diphtheria toxin (DTx) that is considered as a vaccine candidate. Application of CRM(197), together with cholera toxin (CT), onto the tape-stripped skin of mice elicited antibody responses that had anti-DTx neutralizing activity. Vaccine delivery onto mildly ablated skin or intact skin did not elicit any detectable anti-CRM(197) antibodies. Mice immunized with CRM(197) alone onto the tape-stripped skin mounted a vigorous antigen-specific proliferative response. In contrast, the induction of cellular immunity after CRM(197) deposition onto mildly ablated or intact skin was adjuvant dependent. Furthermore, epidermal cells were activated and underwent apoptosis that was more pronounced when the stratum corneum was removed by tape stripping. Overall, these findings highlight the potential for transcutaneous delivery of CRM(197) and establish a correlation between the degree of barrier disruption and levels of antigen-specific immune responses. Moreover, these results provide the first evidence that the development of a transcutaneous immunization strategy for diphtheria, based on simple and practical methods to disrupt the skin barrier, is feasible.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus antitoxin. One laboratory also performed the tetanus ToBI assay. The correlation coefficient (r) between Vero cell assay and ELISA for diphtheria antitoxin ranged from 0.76 to 0.91 in the different laboratories. The correlation between diphtheria serological assays and challenge assays were confirmed satisfactory as ca. 90 per cent of serum-estimates lead to correct prediction of mortality. All laboratories had identical rankings of the vaccines in all serological assays and in the valid challenge assays. The ranking order was identical to assumed/provided potency for the highest and the lowest vaccine. Two of the vaccines had an inversion in some assays and laboratories. As these two vaccines have almost identical potencies in all assays, these inversions are not significant. As the vaccine doses were optimised for the diphtheria component, serum anti-tetanus toxoid/toxin activities varied widely between the vaccines, making it questionable to apply a parallel line model to calculate exact potencies. However, the dose levels used showed a clear regression and good linearity in general. DTaP vaccines containing the IPV component did not always meet the present Ph. Eur. requirements in the serological assays. It should be further investigated in the Phase III study if this is a general feature of such combined vaccines. Preliminary investigations on samples from two laboratories indicate that the neutralising activity of type 1, 2 and 3 polioviruses can also be detected, in a dose-dependent way. Further studies are in progress with serum samples from other laboratories. In the light of results obtained in the first two phases, it is recommended to proceed with Phase III study to investigate reliability of the in vitro assays. In Phase III it will also be further investigated whether the serological assays for D and T components are suitable for the control of the multi-component vaccines currently marketed in Europe.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

Calibration of replacement international standard and European pharmacopoeia biological reference preparation for tetanus toxoid, adsorbed.

Here we report the characterisation of a preparation of tetanus toxoid, adsorbed, and its calibration by 27 laboratories in 19 countries in a joint international collaborative study co-sponsored by World Health Organization (WHO) Expert Committee of Biological Standardization (ECBS) and the European Biological Standardisation Programme of European Directorate for the Quality of Medicines (EDQM), Council of Europe. Calibration was in terms of the Second International Standard (I.S.) for Tetanus Toxoid, Adsorbed, by the established WHO/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 469 IU/ampoule on the basis of its calibration in guinea-pigs and 496 IU/ampoule on the basis of its calibration in mice. Assessment, both within the collaborative study and as part of candidate characterisation, indicated satisfactory stability of the candidate preparation. This study also provided some information on the effect of mouse strain on potency testing of tetanus vaccines. A limited assessment of the impact of the replacement standard on testing of current production batches of vaccines was also carried out by four manufacturers. This study did not directly address the serological approaches to potency testing. However, one laboratory offered data from mouse serology assay, which gave comparable estimates to in vivo mouse bioassay. Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Tetanus Toxoid, Adsorbed (coded 98/552) by the WHO Expert Committee of Biological Standardization (ECBS) in November 2000. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 2) by the Steering Committee of the Biological Standardisation Programme of the EDQM and approved by the European Pharmacopoeia Commission.