Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange

Abstract

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 μg/mL) compared to Bp-WWC (2.61 ± 0.16 μg/mL) and wild type strain (2.2 μg/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 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL) and Bp-WWD (2.31 ± 0.17 μg/mL). Purified PTd from Bp-WWD at 0.8-1.6 μg/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.

Figures

Figure 1

Vectors for the construction of a modified S1 gene into the allelic-exchange vector pSS4245. A: Allelic-exchange element for replacing the S1 gene by a chloramphenicol resistance cassette, inserted between the S1 flanging regions. B: Allelic-exchange element for returning the modified S1 gene into its exact location in the ptx-ptl operon. To obtain the allelic exchange, these vectors were linearized and inserted into pSS4245, which was then introduced into B. pertussis by conjugative transfer from E. coli SM10

Figure 2

Allelic-exchange procedure. A: Double recombination events leading to the replacement of the S1 gene by a chloramphenicol resistance marker. B: Double recombination events leading to the re-insertion of the modified S1 gene in its original location.

Figure 3

Vectors for the insertion of a second copy of the ptx operon into the B. pertussis chromosome. A: The insertion site for a second copy of the ptx operon was selected between two abandoned genes, each carrying two frameshift mutations. B: Allelic-exchange elements used to insert a chloramphenicol marker into the selected site. C: Schematic structure of the ptx operon with its original promoter. The ptx-ptl terminator was cloned and inserted downstream of the S3 gene. This cluster was finally integrated into the SS4245 derivative to replace the chloramphenicol marker and generate the second allelic-exchange event to insert the second copy of the PT structural genes.

Figure 4

Identification of the R9K and E129G mutations in Bp-WWC and Bp-WWD. Raw sequence data around the mutations are shown for strain Bp-WWD that has two copies of the PT structural cluster. The corresponding sequence alignments are shown for B. pertussis Tohama (consensus sequence) and derivatives Bp-WWC and Bp-WWD.

Figure 5

Vectors for the insertion of a second copy of the prn gene into the B. pertussis chromosome. A: The insertion site for a second copy of the prn gene was selected between two abandoned genes carrying frameshift mutations and a deletion. B: Schematic structure of the prn gene under control of fha promoter and flanking with target integration site. C: Schematic structure of the prn gene under control of its own promoter and flanking with target integration site.

Figure 6

CHO-cell clustering test. The cells were grown to near confluence then dilutions of PT were added and the clustering was scored after 2 days. A: 800 ng PT (strain Bp-WWC). B: Control, no PT added. C: 2.6 pg wt PT (strain Tohama) corresponding to the limit of detection. D. 43 pg wt PT (strain Tohama)