Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity

Abstract

The mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD + binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.

Structure and compositional analysis of aluminum oxyhydroxide adsorbed pertussis vaccine

“…The CD spectra for all three samples were recorded to gain comparative knowledge of secondary structure of the protein. The results are consistent with the recently reported crystal structure for gdPT [38] and showed that it is nearly identical to that of PTx. Although gdPT showed presence of monomer and multimer in solution, where PTx had only multimeric species, the X-ray analysis [35] demonstrated that they both consist of five subunits, referred to as S1, S2, S3, S4, and S5 [38] .…”

“…Although gdPT showed presence of monomer and multimer in solution, where PTx had only multimeric species, the X-ray analysis [35] demonstrated that they both consist of five subunits, referred to as S1, S2, S3, S4, and S5 [38] . In addition, hydrogen–deuterium exchange mass spectrometry revealed distal changes in the S2-S5 subunit interactions resulting in tighter packing of B-oligomer and leading to increased thermal stability [38] . The latter is consistent with Tm of ~70 °C as observed by DSC ( Fig.…”

Acellular Pertussis Vaccine Inhibits Bordetella pertussis Clearance from the Nasal Mucosa of Mice

“…Hence, insufficient neutralization of the locally produced PT in mucosal tissue may support the establishment of long-term bacterial colonization. This hypothesis can now be tested with a reformulated aP vaccine containing the genetically detoxified gdPT antigen with superior protective immunogenicity, which has a preserved tertiary structure with fully preserved conformational neutralizing epitopes [ 62 , 63 , 64 , 65 ].…”

“…It will be important to decipher the mechanism responsible, since extended nasopharyngeal colonization was previously observed also in aP-vaccinated and B. pertussis -challenged non-human primates [ 19 ], a model that mimics the natural infection of human upper airways by B. pertussis more faithfully than the mouse nasal clearance model used here. [ 62 ].…”

Application of xCELLigence real-time cell analysis to the microplate assay for pertussis toxin induced clustering in CHO cells

“…To assess the feasibility of the xCELLigence CHO cell assay for samples that contain none or low levels of PT activity, genetically detoxified PT (gdPT) samples, unspiked or spiked with standard PT were evaluated in parallel by xCELLigence and microscopy methods. The gdPT is produced from a genetically engineered B. pertussis strain that contains two genetic modifications within the S1 subunit of the toxin (Arg9Lys and Glu129Gly) that abolish the enzymatic activity of the protein [31,32]. The gdPT sample matrix is equivalent to the pertussis toxin matrix without the toxin activity, which provides the right environment for representative measurements of the PT concentrations in the spiked samples.…”