Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - VAC2VAC (Vaccine lot to Vaccine lot comparison by consistancy testing - Sofia ref.: 115924)
Teaser
The overall objective of the “Vaccine batch to vaccine batch comparison by consistency testing†project (acronym: VAC2VAC) is to demonstrate proof of concept of the consistency approach for batch release testing of established vaccines. This means that animal-free assays ...
Summary
The overall objective of the “Vaccine batch to vaccine batch comparison by consistency testing†project (acronym: VAC2VAC) 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. Hence the name “consistency approachâ€. It covers vaccine potency, safety and animal welfare. Due to the nature of the animal-free assays, the consistency approach also clearly will speed up the release time so that vaccine batches will be available for vaccination much quicker.
Work performed
Physicochemical methods
Mass spectrometry (LC-MS): assays for Leptospira and DTaP vaccines were successfully set up confirming suitability of MS for further analysis. A panel of physicochemical assays was applied to tetanus toxoid (TT) antigens from a number of manufacturers. From this panel, circular dichroism and fluorescence spectroscopy standout as promising candidate tests to assess structural conformation and stability of the TTs. In addition, LC-MS of the toxoid bulks showed that a purity profile can be generated. To facilitate these studies, tailor-made desorption protocols are being developed for human DTaP vaccines. Furthermore, the set up of enzymatic assays simulating antigen degradation by immune cells has started.
Immunochemical methods
Good progress has been made towards the overall objective. For veterinary rabies vaccine, the results obtained do indicate that a glycoprotein ELISA is a viable non-animal approach for potency testing of veterinary rabies vaccines and the industry partners will continue with in-house development of ELISA methods for rabies vaccine and will share progress with the VAC2VAC consortium. For TBEV, very good progress has been made for development of immunoassays for TBEV; Pfizer have joined the consortium and have developed a monoclonal antibody ELISA for TBEV; AGES have developed another ELISA method based on a monoclonal antibody that was selected after extensive characterisation of a panel of TBEV monoclonals; both ELISAs are proposed for a multi-centre transferability study during year 4. For Clostridium chauvoei vaccine, an ELISA format has been developed and appears to be sufficiently sensitive for testing vaccine products. For IBV the characterisation and selection of mAbs is still ongoing.
Cell based assays
To replace the RPT for TBEV vaccine, the monocyte-activation test (MAT) using human peripheral blood mononuclear cells (h-PBMC) was optimized, validated and transferred to the respective industry partner.
The inflammasome activation assay has a biologically relevant read-out that could be useful for characterisation of intermediate alum-containing products. Cell-based assays to study IBV and Leptospira vaccines using antigen-presenting cells (APC) are under development.
Innate immune fingerprinting: The FeLV vaccine component Quil-A was found to induce NF-κB-signaling in a dose-dependent and reproducible manner. For the IBR vaccine, no signal was detected using the entire bioassay library.
Human B-cell assay for consistency testing of DTaP antigens: It could be demonstrated that it is feasible to use PBMC isolated from buffy coats from healthy or freshly vaccinated donors for this ELISpot-based assay.
To assess toxoid processing and subsequent peptide presentation by APC, an in vitro co-culture assay of toxoid-primed human APC and tetanus- or diphtheria toxoid-specific CD4+ T cell hybridoma’s is being set up. In addition, assays are under development to assay T cell activation induced by veterinary IBV and Leptospira vaccines.
In vitro safety test for bulk tetanus toxoid: a detection limit for tetanus toxin as low as 0.03 nM was reached and modifications to assay conditions will be tested to improve sensitivity of the assay and bring it close to what is detectable with in vivo assays. For veterinary C. perfringens C it was found that both A10 and THP-1 cell lines are susceptible to the C. perfringens C non-inactivated antigen in a concentration-dependent manner and with a sensitivity in the required range.
Multiparametric assays and bioinformatics
Characterization of Clostridium tetani seed strains was performed using DNA, RNA and protein analysis. Two DNA-based methods have been established and are ready to enter method validation. A mass spectrometry-based method for protein characterization was successfully set up and validation has started.
The development of an alternative pertussis vaccine safety test was initiated with the goal to obtain a fully quantitative
Final results
Analysis of the production process and in vitro methods optimised or developed during the project provide fundamental understanding of key parameters for antigen integrity, conformation and functionality as well as for antigen-adjuvant interaction. 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.
Website & more info
More info: http://www.vac2vac.eu.