Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation

PLoS One. 2016 Aug 22;11(8):e0161428. doi: 10.1371/journal.pone.0161428. eCollection 2016.


The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and in vivo potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest in vivo potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells in vitro were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the lgmA-C genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring in vitro testing of vaccine quality a step closer.

MeSH terms

  • Antigen Presentation
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology*
  • Biological Assay
  • Bordetella pertussis / drug effects
  • Bordetella pertussis / genetics
  • Bordetella pertussis / immunology*
  • Bordetella pertussis / pathogenicity
  • Carbohydrate Sequence
  • Dendritic Cells / drug effects
  • Dendritic Cells / immunology*
  • Dendritic Cells / microbiology
  • Gene Expression
  • HEK293 Cells
  • Host-Pathogen Interactions
  • Humans
  • Lipopolysaccharides / pharmacology
  • Magnesium Sulfate / pharmacology
  • Monocytes / drug effects
  • Monocytes / immunology*
  • Monocytes / microbiology
  • Pertussis Vaccine / pharmacology*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Primary Cell Culture
  • Toll-Like Receptor 2 / genetics
  • Toll-Like Receptor 2 / immunology
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / immunology*
  • Trans-Activators / genetics
  • Trans-Activators / immunology*
  • Transfection
  • Transgenes
  • Vaccines, Attenuated
  • Virulence Factors / genetics
  • Virulence Factors / immunology
  • Whooping Cough / prevention & control


  • Bacterial Proteins
  • Bvg accessory factor protein, Bordetella pertussis
  • Lipopolysaccharides
  • Pertussis Vaccine
  • TLR2 protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 2
  • Toll-Like Receptor 4
  • Trans-Activators
  • Vaccines, Attenuated
  • Virulence Factors
  • Magnesium Sulfate

Grants and funding

This study was part of the programme Dutch ministry of Health, Welfare and Sports and the Dutch ministry of Economic Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.