Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions.

BACKGROUND: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. METHODS: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. RESULTS: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. CONCLUSIONS: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

Establishment of the national reference standard for tetanus toxoid in Thailand.

The use of reference materials is the basis of standardization and quality control of biologicals such as vaccines produced by different manufacturers and lot-to-lot consistency. The aim of this study was to establish a secondary local and national reference standard of adsorbed tetanus toxoid that can be used for tetanus toxoid vaccine potency testing. Concentrated bulk of tetanus toxoid was adjuvanted and aliquoted before lyophilization. Lyophilized product was tested for biological and physicochemical qualities, including moisture content, identity, appearance, antigen content, degree of adsorption, and sterility. The potency of the candidate reference material was calibrated against the 4th World Health Organization International Standard (WHO IS) for tetanus toxoid by two independent laboratories (BioNet-Asia and Thai National Control Laboratory) using the WHO mouse challenge test. A total of 839 vials with lyophilized tetanus toxoid reference material were produced. Potency was estimated at 115 IU/vial [intra-laboratory geometric coefficient of variation (GCV) of 7 tests was 16.5%] and 112 IU/vial (intra-laboratory GCV of 5 tests was 38.6%) at the two laboratories, with an inter-laboratory GCV of 25.5%. The potency of the candidate standard was assigned a value of 114 IU/vial. The candidate reference standard was approved by The Thai National Central Laboratory (NCL) as the Thai national tetanus toxoid reference standard.

Immunizations for adult women.

Immunizations protect individual persons and contribute to public health by reducing morbidity and mortality associated with common infectious diseases. In this Practice Pearl, we review guidelines for adult immunizations and recent and potential changes in vaccines.

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity.

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the "3R" principles of replacing, reducing and refining animal tests, the "binding and cleavage" (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics. Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit. In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.

Real time and accelerated stability studies of Tetanus toxoid manufactured in public sector facilities of Pakistan.

Tetanus is an acute illness represented by comprehensive increased inflexibility and spastic spasms of skeletal muscles. The poor quality tetanus toxoid vaccine can raise the prevalence of neonatal tetanus. WHO has taken numerous steps to assist national regulatory authorities and vaccine manufacturers to ensure its quality and efficacy. It has formulated international principles for stability evaluation of each vaccine, which are available in the form of recommendations and guidelines. The aim of present study was to ensure the stability of tetanus vaccines produced by National Institute of Health, Islamabad, Pakistan by employing standardized methods to ensure constancy of tetanus toxoid at elevated temperature, if during storage/transportation cold chain may not be maintained in hot weather. A total of three batches filled during full-scale production were tested. All Stability studies determination were performed on final products stored at 2-8°C and elevated temperatures in conformance with the ICH Guideline of Stability Testing of Biological Products. These studies gave comparison between real time shelf-life stability and accelerated stability studies. The findings indicate long-term thermo stability and prove that this tetanus vaccine can remain efficient under setting of routine use when suggested measures for storage and handling are followed in true spirit.

Binding and cleavage (BINACLE) assay for the functional in vitro detection of tetanus toxin: applicability as alternative method for the safety testing of tetanus toxoids during vaccine production.

Tetanus toxoids (i.e. chemically inactivated preparations of tetanus neurotoxin) are used for the production of tetanus vaccines. In order to exclude the risk of residual toxicity or of a "reversion to toxicity", each batch of tetanus toxoid is subject to strict safety testing. Up to now, these prescribed safety tests have to be performed as in vivo toxicity tests in guinea pigs. However, as animal tests are generally slow, costly and ethically disputable, a replacement by an in vitro method would be desirable. A suitable alternative method would have to be able to sensitively detect already low concentrations of active tetanus neurotoxin in matrices containing large amounts of inactivated toxoid molecules. We have developed a method which detects active tetanus neurotoxin molecules based on their specific receptor-binding capacity as well as their proteolytic activity. By taking into account two relevant functional characteristics, this combined "BINding And CLEavage" (BINACLE) assay more reliably discriminates between toxic and detoxified molecules than other in vitro assays which solely rely on one single toxin function (e.g. endopeptidase assays). Data from an in-house validation show that the BINACLE assay is able to detect active tetanus neurotoxin with a detection limit comparable to the in vivo test. The sensitive detection of active toxin which has been spiked into toxoid samples from different manufacturers could also be demonstrated. Specificity and precision of the method have been shown to be satisfactory. The presented data indicate that for toxoid batches from some of the most relevant European vaccine manufacturers, the BINACLE assay may represent a potential alternative to the prescribed animal safety tests. In addition, this novel method may also provide a convenient tool for monitoring batch-to-batch consistency during toxoid production.

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 Tetanus Toxoid Adsorbed.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.

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.

Human papillomavirus vaccination rates and state mandates for tetanus-containing vaccines.

OBJECTIVE: We sought to examine nationally the association between school mandates for adolescent tetanus-containing vaccines (Td and/or Tdap) and adolescent female human papillomavirus (HPV) vaccination. METHODS: Each state was categorized by whether a school mandate for adolescent Td and/or Tdap vaccines was enacted. Mean HPV vaccine series initiation levels among adolescent females were compared between each mandate category. RESULTS: Mean HPV vaccine series initiation levels were significantly lower in states without Td/Tdap vaccine mandates than in those with mandates (42.9% vs. 47.3%; p=0.004). CONCLUSIONS: School mandates for adolescent Td/Tdap vaccination may have a carry-over effect on HPV vaccination.

Accelerated stability studies for moisture-induced aggregation of tetanus toxoid.

PURPOSE: The study was carried out to evaluate the effect of exposing solid tetanus toxoid to moisture in two different ways on the structure and function of the toxoid. METHODS: Tetanus toxoid was exposed to moisture by (i) the addition of an optimized amount of buffer and (ii) incubation under an environment provided by a saturated solution of K(2)CrO(4.) The changes in the conformational, structural and antigenic properties of tetanus toxoid were measured and compared. RESULTS: Results show that even at a similar level of moisture-induced aggregation, the amounts of water absorbed by the two preparations of tetanus toxoid are different. Differences in antigenicity and changes in structure of the toxoid at primary, secondary and tertiary structure levels were seen. CONCLUSION: Although both conditions are used to mimic accelerated stability conditions in the laboratory, the final products are different in the two cases. Thus, conditions for 'accelerated stability studies' for therapeutic proteins need to be selected with care so that they resemble the fate of the actual product.

Collaborative study for the validation of alternative in vitro potency assays for human tetanus immunoglobulins.

An international collaborative study to validate 2 alternative in vitro methods for the potency testing of human tetanus immunoglobulin products was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). The study, run in the framework of the Biological Standardisation Programme (BSP) under the aegis of the European Commission and the Council of Europe, involved 21 official medicines control and industry laboratories from 15 countries. Both methods, an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA), showed good reproducibility, repeatability and precision. EIA and TIA discriminated between low, medium and high potency samples. Potency estimates correlated well and both values were in close agreement with those obtained by in vivo methods. Moreover, these alternative methods allowed to resolve discrepant results between laboratories that were due to product potency loss and reporting errors. The study demonstrated that EIA and TIA are suitable quality control methods for tetanus immunoglobulin, which can be standardised in a control laboratory using a quality assurance system. Consequently, the Group of Experts on Human Blood and Blood Products of the European Pharmacopoeia revised the monograph on human tetanus immunoglobulins to include both the methods as compendial alternatives to the in vivo mouse challenge assay.

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.

Collaborative study for the validation of alternative in vitro potency assays for human tetanus immunoglobulin.

The European Pharmacopoeia (Ph. Eur.) monograph Human tetanus immunoglobulin (0398) gives a clear outline of the in vivo assay to be performed to determine the potency of human tetanus immunoglobulins during their development. Furthermore, it states that an in vitro method shall be validated for the batch potency estimation. Since no further guidance is given on the in vitro assay, every control laboratory concerned is free to design and validate an in-house method. At the moment there is no agreed in vitro method available. The aim of this study was to validate and compare 2 alternative in vitro assays, i.e. an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA), through an international collaborative study, in view of their eventual inclusion into the Ph. Eur.. The study was run in the framework of the Biological Standardisation Programme (BSP), under the aegis of the European Commission and the Council of Europe. The collaborative study reported here involved 21 laboratories (public and industry) from 15 countries. Initially, 3 samples with low, medium and high potencies were tested by EIA and TIA. Results showed good reproducibility and repeatability of the 2 in vitro methods. The correlation of the data with the in vivo potency assigned by the manufacturers however appeared initially poor for high potency samples. Thorough re-examination of the data showed that the in vivo potencies assigned by the manufacturers had to be corrected: one for potency loss at the time of in vitro testing and one because of a reporting error. After these corrections the values obtained by in vivo and in vitro methods were in close agreement. A supplementary collaborative work was carried out to validate the 2 methods for immunoglobulin products with high potencies. Eight laboratories (public and industry) took part in this additional study to test 3 samples with medium and high potencies by EIA and TIA. Results confirmed that the 2 alternative methods are comparable in terms of assay repeatability, precision and reproducibility. In all laboratories, both methods discriminated between the low, medium and high potency samples. Analysis of the data collected in this study showed a good correlation between EIA and TIA potency estimates as well as a close agreement between values obtained by in vitro and in vivo methods. The study demonstrated that EIA and TIA are suitable quality control methods for polyclonal human tetanus immunoglobulin, which can be standardised in a quality control laboratory using a quality assurance system. Consequently, the Ph. Eur. Group of Experts 6B on Human Blood and Blood products decided in April 2009 to include both methods as examples in the Ph. Eur. monograph 0398 on Human Tetanus immunoglobulin.

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.