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Vaccine lot to Vaccine lot comparison by consistency testing.

Periodic Reporting for period 4 - VAC2VAC (Vaccine lot to Vaccine lot comparison by consistency testing.)

Reporting period: 2019-03-01 to 2020-02-29

The overall objective of the “Vaccine batch to vaccine batch comparison by consistency testing” project is to demonstrate proof of concept of the consistency approach for batch release testing of established vaccines. This means that animal-free assays - instead of animal tests - shall be used to ensure that each vaccine batch produced is consistent with a batch already proven to be safe and efficacious in registration studies.
Physicochemical methods
Antigens (Ag) present in a vaccine are key to the induction of a protective immune response against a specific disease. Therefore, we aim at establishing a set of physicochemical methods to determine the shape and quantitative profile of vaccine antigens.
Mass spectrometry (MS) assays for Leptospira and Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccines were set up and optimized. For Leptospira a semi-quantitative MS method was established laying the basis for the quantitative approach to follow. For DTaP vaccines, very good reproducibility could be obtained in quantifying Ag contained in aluminum hydroxide-adjuvanted, hexavalent vaccines.
A panel of physicochemical assays was applied to tetanus toxoid (TT) Ag, from which circular dichroism (CD) and fluorescence spectroscopy standout as promising candidate tests to assess structural conformation and stability of the TTs. CD revealed the applicability of the higher order structure analysis for pre-adjuvanted TT as well as final adjuvanted products.
Enzymatic assays simulating antigen degradation by immune cells have been set up for all DTaP Ag but Fim2/3. Antigenicity assessment of individual DTaP antigens via biosensor technology has started.
Tailor-made desorption protocols have been developed for human DTaP vaccines, facilitating the use of physicochemical, immunochemical, and cell-based assays

Immunochemical methods
The quantity and quality of vaccine Ag can be determined through immunochemical methods, such as the enzyme-immunosorbent assay (ELISA), what could ensure product safety and effectiveness and the replacement of animal tests for the determination of vaccine potency.
Very good progress has been made in the development of ELISAs for tick-borne encephalitis virus (TBEV). ELISA tests developed by Pfizer and AGES are proposed for a multi-centre transferability study during year 5. For veterinary rabies vaccine, the industry partners will continue with in-house development of ELISA methods and share progress with the consortium. For DTaP vaccine, Multiplex immunoassays are being developed for acellular pertussis antigens together with Luminex assays for D and T. For Clostridium chauvoei vaccine, an ELISA format has been developed and appears to be sensitive for testing vaccine products. For IBV the method development work has been stopped after results made it clear that it was not be possible to produce mAb capable of detecting the IBV antigen in the vaccine.

Cell based assays
Vaccine-induced immunity could be measured in vitro instead of in vivo by using single types or combinations of immune cells, such as human peripheral mononuclear cells (PBMC) T- or B-cells.
A monocyte-activation test using human PBMC was validated and transferred to an industry partner, who adapted the assay to GMP standards and submitted a variation for the implementation of the method in routine quality control. Furthermore, novel biomarkers for potency testing of a TBEV vaccine are being identified.
An inflammasome activation assay was developed. The assay has a biologically relevant read-out that could be useful for characterisation of intermediate alum-containing products. Progress was made in the development of assays that assess T-cell activation induced by IBV and Leptospira vaccines.
Using an innate immune fingerprinting approach, the FeLV vaccine component Quil-A was found to induce NF-κB-signaling in a dose-dependent and reproducible manner.
A B-cell assay based on human PBMC was set up for consistency testing of DTaP antigens. Proof of concept and specificity of the assay could be shown for adsorbed tetanus toxoid.
Work related to the development of a TT- or DT-specific CD4+ T cell hybridoma assay and an in vitro safety test for bulk TT using engineered cell lines has been discontinued due to the impossibility to activated the T cell hybridoma or low sensitivity for replacement of animal test, respectively. For veterinary C. perfring
Implementation of the consistency approach will lead to replacement, reduction or refinement of animal use and could lead to a revision of the monographs for some vaccines. The consistency approach also clearly will speed up the release time so that vaccine batches will be available for vaccination much quicker.
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