A next-generation sequencing based method for determining genetic stability in Clostridium tetani vaccine strains.

Production of tetanus and other clostridial vaccines highly depends on the stable and reproducible production of high toxin levels. This creates a need to ensure the genetic stability of seed strains. We developed a two-stage method for improved assessment of the genetic stability of Clostridium seed strains. This method is based on next-generation sequencing (NGS) of strain DNA and mapping the sequence reads to a reference sequence. The output allows analysis of global genome consistency followed, if necessary, by detailed expert judgement of potential deviations at the gene level. The limit of detection of our method is an order of magnitude better than that of the currently established pulsed-field gel electrophoresis (PFGE). Improved genetic characterization of bacterial seed lots will have a positive impact on the characterization of the production process. This will be a first step towards applying the consistency approach to vaccine batch release of established vaccines. This can contribute to the reduction and ultimately replacement of routinely used animal tests in vaccine production. This work was carried out as part of the Innovative Medicines Initiative 2 (IMI2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing).

Recommendations of the VAC2VAC workshop on the design of multi-centre validation studies.

Within the Innovative Medicines Initiative 2 (IMI 2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing), a workshop has been organised to discuss ways of improving the design of multi-centre validation studies and use the data generated for product-specific validation purposes. Moreover, aspects of validation within the consistency approach context were addressed. This report summarises the discussions and outlines the conclusions and recommendations agreed on by the workshop participants.

Soil organic matter widens the range of water contents for tillage.

The effects of soil organic matter on the water contents for tillage were investigated by sampling soils with a uniform texture, but a range of soil organic carbon (SOC) from two long-term field experiments at Highfield in Rothamsted Research, UK and Askov Experimental Station, Denmark. The treatments studied in Highfield were Bare fallow (BF), Continuous arable rotation (A), Ley-arable (LA) and Grass (G); and in Askov: unfertilized (UNF), ½ mineral fertilizer (½ NPK), 1 mineral fertilizer (1NPK), and 1½ animal manure (1½AM). Minimally disturbed soil cores (100 cm3) were sampled per plot in both locations from 6 to 10 cm depth to generate water retention data. Soil blocks were also sampled at 6-15 cm depth to determine basic soil properties and to measure soil aggregate strength parameters. The range of soil water contents appropriate for tillage were determined using the water retention and the consistency approaches. SOC content in Highfield was in the order: G > LA = A > BF, and in Askov: 1½ AM > 1NPK = ½NPK > UNF. Results showed that different long-term management of the silt loam Highfield soil, and fertilization of the sandy loam Askov soil affected the mechanical properties of the soils- for Highfield soil, aggregates from the G treatment were stronger in terms of rupture energy when wet (-100 hPa matric potential) than the BF treatment. As the soil dried (-300 and -1000 hPa matric potentials), soil aggregates from the G treatment were relatively weaker and more elastic than the BF soil. Our study showed, for both Highfield and Askov soils, a strong positive linear increase in the range of water contents for tillage with increasing contents of SOC. This suggests that management practices leading to increased SOC can improve soil workability by increasing the range of water contents for tillage. We recommended using the consistency approach over the water retention approach for determining the range of water contents for tillage because it seems to give realistic estimates of the water contents for tillage.

Drivers and barriers in the consistency approach for vaccine batch release testing: Report of an international workshop.

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine, and the principle that the quality of subsequent batches is the consequence of the application of consistent production of batches monitored by a GMP quality system. Efforts to implement the consistency approach are supported by several drivers from industry, government, and research, but there are also several barriers that must be overcome. A workshop entitled "Consistency Approach, Drivers and Barriers" was organized, which aimed to discuss and identify drivers and barriers for the implementation of the 3Rs in the consistency approach from three different perspectives/domains (industry, regulatory and science frameworks). The workshop contributed to a better understanding of these drivers and barriers and resulted in recommendations to improve the overall regulatory processes for the consistency approach. With this report, we summarise the outcome of this workshop and intend to offer a constructive contribution to the international discussion on regulatory acceptance of the consistency approach.

In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.

Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in diphtheria-tetanus based vaccines and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity, and is suitable for detecting DTxd in a range of different human vaccine products. We demonstrate the ability of the assay to discriminate between batches of different content and quality using vaccine batches that were prepared to contain differing amounts of DTxd or were altered by exposure to heat or oxidative stress. We also demonstrate successful transfer of the method to other laboratories and show that different diphtheria antigen materials may be able to serve as a reference antigen for local standardization of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Diphtheria vaccines help to protect against diphtheria infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of diphtheria vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different diphtheria vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and recognized by regulators, this assay will greatly reduce the number of animals needed for batch release of diphtheria vaccines.

Development of a monoclonal antibody sandwich ELISA for the quality control of human and animal tetanus vaccines.

Antigen identity, quantity and integrity are key factors to be evaluated as part of consistency testing of tetanus vaccines. Here we have developed a monoclonal antibody sandwich ELISA to measure the relative amount and quality of tetanus toxoid (TTxd) in human and animal tetanus vaccines. The ELISA is highly specific, has good dilutional linearity, and is suitable for detecting TTxd in a range of different products. We have demonstrated the ability of the assay to discriminate between batches of different content, using vaccine batches that had been prepared to contain differing amounts of TTxd, and of different quality, using samples of non-adjuvanted TTxd that had been exposed to sonication and final lot vaccines that had been exposed to heat or oxidative stress. We have also demonstrated successful transfer of the method to other laboratories and have shown that different tetanus antigen materials may be able to serve as a reference antigen for standardization of the method. The results show this test has the potential to play a key role in a control strategy no longer including an in vivo potency test.

Tetanus vaccines help to protect against tetanus infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of tetanus vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different tetanus vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and accepted by regulators, this assay will greatly reduce the number of animals needed for batch release of tetanus vaccines.

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, the introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is challenging. AREAS COVERED: This report describes the hurdles encountered in substituting in vivo assays and ways to overcome these and provides arguments why more advanced in vitro alternatives are superior, not only as a 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 in vivo batch release test if an appropriate non-animal testing strategy is 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 expect 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 related 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 regions.

Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation.

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine to identify critical quality attributes that inform the use of a comprehensive set of non-animal tests to release the vaccine, together with the principle that the quality of subsequent batches follows from their consistent production. Many vaccine antigens are by themselves not able to induce a protective immune response. The antigens are therefore administered together with adjuvant, most often by adsorption to aluminium salts. Adjuvant function is an important component of vaccine potency, and an important quality attribute of the final product. Aluminium adjuvants are capable of inducing NLRP3 inflammasome activation. The aim of this study was to develop and evaluate an in vitro assay for NLRP3 inflammasome activation by aluminium-adjuvanted vaccines. We evaluated the effects of Diphtheria-Tetanus-acellular Pertussis combination vaccines from two manufacturers and their respective adjuvants, aluminium phosphate (AP) and aluminium hydroxide (AH), in an in vitro assay for NLRP3 inflammasome activation. All vaccines and adjuvants tested showed a dose-dependent increase in IL-1ß production and a concomitant decrease in cell viability, suggesting NLRP3 inflammasome activation. The results were analysed by benchmark dose modelling, showing a similar 50% effective dose (ED50) for the two vaccine batches and corresponding adjuvant of manufacturer A (AP), and a similar ED50 for the two vaccine batches and corresponding adjuvant of manufacturer B (AH). This suggests that NLRP3 inflammasome activation is determined by the adjuvant only. Repeated freeze-thaw cycles reduced the adjuvant biological activity of AH, but not AP. Inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterization of the adjuvant.

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.

Development and validation of a targeted LC-MS/MS quantitation method to monitor cell culture expression of tetanus neurotoxin during vaccine production.

The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20% mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored.