Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions.

Jeroen L A Pennings,Raphaël Esson,Eric Abachin,Jean-Baptiste Claude,Céline Vanhee,Hennie Hodemaekers,Sylvie Uhlrich,Rob J Vandebriel,Antoine Francotte

doi:10.3390/toxins14010031

Abstract

Background: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. Methods: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. Results: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. Conclusions: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

Highlights

The detoxified form of the Clostridium tetani neurotoxin (TeNT) is the essential component of tetanus vaccines
The manufacturing process starts with the generation of stable and good manufacturing practice (GMP)-monitored toxin-producing C. tetani seed lots, which are cultivated for days until the end of their growth phase
The goal of this study is to identify RNA signatures related to consistent TeNT production during batch fermentation under manufacturing conditions, with a view to better monitoring the consistency of this manufacturing step

Summary

Introduction

The detoxified form of the Clostridium tetani neurotoxin (TeNT) is the essential component of (human) tetanus vaccines. The manufacturing process starts with the generation of stable and good manufacturing practice (GMP)-monitored toxin-producing C. tetani seed lots, which are cultivated for days until the end of their growth phase. At this point, TeNT is harvested from the culture supernatant, purified, and inactivated by the addition of formaldehyde. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing

Objectives

Methods

Results