Development of an Approach for the Laboratory Toxicological Evaluation ofBordetella PertussisAdenylate Cyclase Genetic Toxoid Constructs as Multipurpose Vaccine

Abstract

Detoxified recombinant CyaA constructs have been developed as potential viral and tumoral epitope carriers for immunoprophylactic and therapeutic vaccination and as antigen candidates for inclusion in acellular pertussis vaccines. In this work, we attempt to explore a test system for the laboratory safety evaluation of CyaA preparations. Endotoxin was determined in vitro by the Limulus amoebocyte lysate assay. Cytotoxicity was measured by a tetrazolium salt test and a lactate dehydrogenase release assay using murine and human phagocytic cell lines. Cell viability was < 50% at concentrations > 1 µg/mL of the wild type toxin and concentrations in the nanogram range inhibited the zymosan-induced oxidative burst as measured by chemiluminescence. However, no effects were observed for detoxified and non-acylated forms of CyaA at comparable and higher toxin concentrations. Effects found in the in vitro assays could not be related to the in vivo mouse weight gain test used as a general toxicity test. In vitro cytotoxicity and oxidative burst studies on phagocytes, and the evaluation of endotoxin levels are useful for safety screening of CyaA constructs. However, in vivo specific toxicity tests and potential toxic implications of enzymatic activity-independent effects of CyaA on cell function should be investigated. ABBREVIATIONSCyaA, adenylate cyclase toxin; MHC, major histocompatibility complex; CTL, cytotoxic T lymphocyte; cAMP, cyclic adenosine monophosphate; CyaA-wt, wild type adenylate cyclase toxin; CyaA-E5, detoxified adenylate cyclase toxin; proCyaA-wt, wild type adenylate cyclase protoxin; proCyaA-E5, detoxified adenylate cyclase protoxin; CyaA-E5-Tyros, detoxified adenylate cyclase toxin with tyrosinase epitope

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

“…Indeed, the cytotoxic action of enzymatically active CyaA on CD11b + phagocytes was documented repeatedly at doses lower than 10 ng/ml, where less than 1 ng/ml of active CyaA toxin quantitatively inhibits oxidative burst in neutrophils [32]. As low CyaA doses as 5 to 10 ng/ml elicit monocyte ruffling and a near-instant inhibition of CR3-mediated opsonophagocytosis or arrest of the fluid-phase uptake, respectively.…”

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

“…cytic killing capacities of neutrophils and macrophages (6)(7)(8)(9)(10). With efficacy of about 2 orders of magnitude lower, the CyaA toxin can also penetrate non-myeloid cells that lack the CR3 receptor (CD11b Ϫ cells), where due to its extremely active AC enzyme it can elevate cAMP concentrations to well detectable and physiologically relevant concentrations in epithelial and other host cells (10,11).…”

Rapid Purification of Endotoxin-Free RTX Toxins

“…Previously, several methods for reduction of LPS contamination of purified CyaA toxin preparations were devised, including extensive washing of toxin-containing inclusion bodies with buffers containing 2 M urea or 1% ( v / v ) N-octyl-glucoside [23], removal of LPS from phenyl-sepharose-bound CyaA by 60% ( v / v ) isopropanol washes [24,25,26] or re-chromatography of purified CyaA on hydroxyapatite [27]. However, these purification procedures are laborious and typically reduce the yield or the specific activity of the purified toxin.…”