Pertussis vaccine testing
2003 – 2007 Dr Hadwen Trust Research Grant
Dr D Xing, National Institute for Biological Standards and Control, Potters Bar, Herts.
Identification of gene markers for in vitro toxicity control test for pertussis vaccines.
Dr Dorothy Xing is a Principal Scientist in the Division of Bacteriology at the NIBSC in Hertfordshire. She has a background in microbiology and pharmacy, and her recent work has focused on improvements in standardisation and safety testing of pertussis vaccines.
Pertussis vaccines are widely used to prevent whooping cough, a highly contagious and life-threatening disease caused by the bacterium Bordetella pertussis. A major constituent of the vaccines is a detoxified form of pertussis toxin. Regulatory authorities require each batch of vaccine be checked for residual toxicity or reversion to toxicity, with animal tests.
The mouse weight gain test (MWGT) and the Histamine Sensitisation Test (HIST), a lethal test in mice, are used in the quality control of pertussis vaccines. The precise mechanisms underlying these tests and the toxicological correlations between humans and animals are not clear. In addition, variations in the performance of HIST frequently lead to repeat testing and large numbers of mice are used. More robust and accurate in vitro tests are needed to replace the animal tests [1, 2].
The Dr Hadwen Trust supported Dr Dorothy Xing’s research to find more accurate in vitro assays to replace the routine animal tests. By studying changes in gene expression in selected human cell lines exposed to pertussis toxin, Dr Xing aimed to identify gene markers of toxicity.
Initial experiments with microarray technology examined gene expression profiles of primary HUVEC (human umbilical vein endothelial cells) and the NL20 cell line (human bronchial epithelial cells) after pertussis toxin treatment in comparison with untreated controls. NL20 cells represent the site of infection and HUVEC can provide important information on vascular permeability.
Further experiments focused on using quantitative RT-PCR with HUVEC cells and found increased expression of genes involved in vessel tone and vascular permissibility, apoptosis, cell death and cell injury. Certain genes involved in cell protection and anti-apoptosis were down-regulated after pertussis toxin treatment.
B. pertussis infection can be accompanied by severe neurological disorders, and it has been speculated that B. pertussis may increase cerebral vascular permeability resulting in encephalopathy. It could be that pertussis toxin affects endothelial cells, and this study found changes in the expression of genes involved in permissibility and vessel tone.
Expression of the adhesion molecule gene ICAM-1 was up-regulated, as were genes involved in the regulation of blood pressure and angiotensin system. A subsequent bioimmunoassay established dose response changes of ICAM-1 by HUVEC after incubation with pertussis toxin, with TNF-alpha and histamine. The addition of TNF-alpha made HUVEC more susceptible to the effects of pertussis toxin and enhanced the assay sensitivity.
Functional assays confirmed the results obtained by the gene expression experiments: that pertussis toxin could increase cell permeability and induce endothelial cell migration, possibly by modifying angiogenesis.
Results of this study showed that ICAM-1 might serve as a gene marker of pertussis toxicity and the simple immunoassay method used to determine ICAM-1 at the protein level has the potential to be a reliable non-animal method for determining pertussis toxicity. The results also indicated that a permeability assay with endothelial cell monolayers has potential as an alternative to the animal tests.
At this stage, further validation of the immunobioassay and permeability assay is still needed, but the final goal is the implementation of these assays into the vaccine regulatory control requirements to replace animal tests.
Summary
- Quality control of batches of pertussis vaccines involves animal testing.
- Current animal tests on mice are imprecise and need replacement.
- Microarray studies were conducted of human cells treated with pertussis toxin to identify potential gene markers of toxicity.
- Microarrays detected changes in the expression of more than 100 genes in response to pertussis toxin.
- RT-PCR of human umbilical vein endothelial cells (HUVEC) confirmed changes in the expression of genes involved in vessel tone and vascular permissibility, apoptosis and cell death in response to pertussis toxin.
- Pertussis toxin induces changes in cell permeability and migration of endothelial cells and consequently may play a role in angiogenesis.
- ICAM-1 was identified as a potential marker of pertussis toxicity in a bioimmunoassay with HUVEC cells. This assay is potentially an alternative to the animal (HIST) test.
- An in vitro permeability assay also has potential to replace animal tests.
- At present, both assays will need further validation before implementation.
References
1. van Straaten-van de Kappelle I, van der Gun JW, Marsman FR, Hendriksen CF, van de Donk HJ (1997). Collaborative study on test systems to assess toxicity of whole cell pertussis vaccine. Biologicals 25:41-57.
2. Corbel MJ & Xing DKL (2004). Toxicity and potency evaluation of pertussis vaccines. Expert Rev Vaccines 3:89-101.