New method to detect safety and potency of whooping cough vaccines

Project acronym: Pertussay

Objectives: Develop a mechanism-based in vitro alternative to replace the mouse histamine sensitization test to detect residual pertussis toxin in pertussis vaccines.

Key collaborations: National Institute for Public Health and the Environment (RIVM)

Funding: National Institute for Public Health and the Environment (RIVM)

Project group: C. Krul, PhD (project leader within HU, advice), S. Vaessen, PhD (scientist, experimental work and reporting), S. Verkoeijen, PhD (scientist, experimental work and reporting)

External partners: A. Akkermans, BSc (RIVM, overall project leader), R. Vandebriel, PhD (RIVM, advice), M. Bruysters, PhD (RIVM, advice), J. Pennings, PhD (RIVM, biostatician)

Relevant publications:

1. Vaessen S, Verkoeijen S, Vandebriel R, Bruysters M, Pennings J, Bos R, Krul C, Akkermans A (2013). Identification of biomarkers to detect residual pertussis toxin using microarray analysis of human dendritic cells. In Press, Vaccine

2. Bache C, Hoonakker M, Hendriksen C, Buchheit KH, Spreitzer I, Montag T. (2012) Workshop on Animal free Detection of Pertussis Toxin in Vaccines-Alternatives to the Histamine Sensitisation Test. Biologicals 40:309.

3. Locht C, Coutte L, Mielcarek N (2011). The ins and outs of pertussis toxin. FEBS J 278(23):4668-82.

4. Mattoo S, Cherry JD (2005). Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18(2):326-82.

Authors: Dr. Stefan Vaessen and
Dr. Saertje Verkoeijen

Part of the text and art-work of this article has been produced by
Tessa Louwerens Tekst & Beeld

Main aims and objectives

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the main antigens used in pertussis vaccines. Although pertussis vaccines are routinely administered to infants to protect them from whooping cough, an adequate safety test for pertussis toxin (PTx) is not available. The mouse histamine sensitization test (HIST) is currently the only assay accepted by regulatory authorities to test for the absence of active PTx in vaccines. However, this is a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e. ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PTx. Thus, an alternative for the HIST is highly desirable. In this project we aimed to develop an alternative in vitro test based on the adverse biological effects of PTx in humans.

Materials and Methods

We used microarray gene expression analysis to find genes that are specifically up- or downregulated by PTx. This technique allows simultaneous characterization of all genes that are affected by PTx in one analysis. Using this approach we identified six genes in human immature monocyte-derived dendritic cells (a relevant cell type for in vivo PTx effects) that were specifically upregulated by PTx and can be used as biomarkers to detect presence of PTx during the production of vaccines. These results have been published in the scientific journal Vaccine (Vaessen et al., 2013; In Press).

This project is an example of how the increased understanding of human biology can contribute to the development of relevant and mechanism-based safety tests. Many safety tests used today were developed a long time ago, when our understanding of (human) biology was limited. These tests were often based on coincidental observations and their relevance and underlying mechanisms are mostly unknown.  Focusing on the biological effects of PTx in the human body, human cell types involved in these in vivo effects of PTx were selected for study to ensure the biological relevance of the biomarkers that we discovered in this project.

Collaboration and education

The project is a collaboration between the National Institute for Public Health and the Environment (RIVM) and the University of Applied Sciences Utrecht. In this collaboration different fields of expertise are represented, including theoretical expertise in the (pertussis) vaccine field, experimental expertise in the development of in vitro assays, knowledge of regulatory affairs and biostatistical expertise, to optimize the outcome of the project.  The Pertussay project is used as a topic in several lectures (e.g. in the Minor ‘Food and Pharma’). Also, students can participate in this project during the minor ‘Food and Pharma’ or during other phases of their training and work with us in the laboratory.

Benefits and applications with respect to alternatives to animal testing

The final contribution to replacement of animal testing depends largely on the outcome of this project in the future.  Experimentally we face challenges in the limited sensitivity of the biomarkers in combination with toxicity of the aluminum adjuvants that is present in vaccine end products. Right now, with limited sensitivity of the assay and lack of a solution for the problem regarding toxicity of the adjuvans, the assay is only fit to replace animal testing of intermediate products. However, if we succeed in solving the mentioned problems, the impact will be much greater.

We are currently working on decreasing the toxic effects of the adjuvans to allow products to be tested in a more concentrated way, thereby increasing the sensitivity and improving the limit of detection. We will also explore the possibilities of using other cell lines that are potentially more sensitive to PTx and find biomarkers using the same approach.  Further development of assays using these biomarkers might eventually result in detection assays that are accepted by regulatory authorities and can replace the HIST for vaccine end products as well.

Furthermore, even though the project focused specifically on finding functional biomarkers to detect the presence of PTx in pertussis vaccines, the approach could be an example for many other safety tests to develop better alternatives.