Dataset Information

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.

SUBMITTER: Ausar SF

PROVIDER: S-EPMC7406505 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Ausar Salvador F SF Zhu Shaolong S Duprez Jessica J Cohen Michael M Bertrand Thomas T Steier Valérie V Wilson Derek J DJ Li Stephen S Sheung Anthony A Brookes Roger H RH Pedyczak Artur A Rak Alexey A Andrew James D D

Communications biology 20200805 1

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 impa ...[more]

PMID: 32759959

Similar Datasets

Project description:BACKGROUND:Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN. RESULTS:Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 ?g/mL) compared to Bp-WWC (2.61 ± 0.16 ?g/mL) and wild type strain (2.2 ?g/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 ?g/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 ?g/mL) and Bp-WWD (2.31 ± 0.17 ?g/mL). Purified PTd from Bp-WWD at 0.8-1.6 ?g/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well. CONCLUSIONS:We have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets