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Animal-free Drug Testing – Cell-based in Vitro Assay for Laboratory Efficacy Testing of Tetanus Toxoid Vaccines

11 / 2021

Batch testing of tetanus toxoid vaccines is currently based on in vivo models, i.e. animal testing. In the interests of animal welfare, there is a need here to replace the established process with an innovative in vitro method. Scientists from Paul-Ehrlich-Institut (PEI) have now managed to develop a cell-based in vitro test for experimental testing of tetanus toxoid vaccines. The researchers report on this work in the Nature Partner Journal "npj / Vaccines".

Clostridium Tetani Bacteria (Source: nobeastofierce/shutterstock.com)

Vaccines provide protection against infectious diseases such as poliomyelitis, measles, diphtheria and tetanus. Since we started immunising infants against these pathogens and the toxins that cause bacterial infectious diseases, global rates of infection have fallen significantly. This has led to a drop in infant and child mortality rates worldwide.

Nevertheless, it remains essential to test each batch of vaccines for quality and properties relating to efficacy in a potency test. The European Pharmacopoeia still prescribes animal experiments (in vivo assays) for many of the required tests. Paul-Ehrlich-Institut has long been committed to minimising the use of animals in experimental drug testing – without any impairment of quality, safety and efficacy – and has been developing alternative methods for many years.

Tetanus is a serious disease caused by infection with the Clostridium tetani bacterium via a wound. The bacterial toxins attack the nervous system, so an infection leads to painful muscle spasms and ultimately to death. In the production of the authorised vaccines against tetanus, the toxin is inactivated by means of formaldehyde. The resulting tetanus toxoid is the protective antigen in the tetanus toxoid vaccine and is harmless to humans. In order to ensure the efficacy of the vaccines by means of a potency test, testing based on animal experiments – the so-called in vivo potency assays – have been prescribed in the pharmacopoeia for decades.

The aim of the experiments carried out by researchers at the Paul-Ehrlich-Institut is to develop a reliable in vitro potency assay for tetanus toxoid vaccines so that it can replace animal testing in future. The team working under Professor Dr Isabelle Bekeredjian-Ding, head of the Microbiology section at Paul-Ehrlich-Institut, and Dr Olga Tichá, the lead author, used knowledge of the mechanism with which the immune system prompts an immune response against tetanus toxoid. As part of the process, human peripheral blood leukocytes were stimulated with tetanus toxoid and low concentrations of a toll-like receptor ligand. After five days the immune response was quantified based on the production of the tetanus toxoid-specific immunoglobin G (IgG) secreted by the leukocytes. Proof of feasibility was provided with paired samples from donors before and after vaccination. With the use of peripheral blood leukocytes from buffy coats, a by-product created in the manufacturing of blood products, the specific reaction to tetanus toxoid could be reproduced in 30% of the donated cell preparations. The level of tetanus toxoid-induced IgG production correlated with a higher number of memory B-cells in the leukocyte donations, the proportion of which will in future be used for selection of buffy coats suitable for the in vitro potency assay. It was also shown that tetanus toxoid-specific immunoglobulin G was also detectable when the blood leukocytes were stimulated with the active agents of a DTaP combination vaccine (diphtheria, tetanus, acellular pertussis).

"This test offers a practicable way of verifying the specific immune response through detection of the B-cell differentiation and the secretion of tetanus toxoid-specific immunoglobulin G by vaccine antigen and complete vaccine," was Bekeredjian-Ding's conclusion on the study. In the future, the functional assay can be used with physicochemical and immunochemical methods to replace the animal testing still currently prescribed for batch testing of tetanus toxoid-containing vaccines. This would be another important milestone on the path towards animal-free drug testing.

Original Publication

Tichá O, Klemm D, Moos L, Bekeredjian-Ding I (2021): A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen.
NPJ Vaccines 6: 88.
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Updated: 06.07.2021