title: 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.
authors.affiliation: Department of Biotechnology, Mahidol University, 272 Rama 6 Road, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Wasin
authors.initials: W
authors.lastname: Buasri
authors.forename: Attawut
authors.initials: A
authors.lastname: Impoolsup
authors.forename: Chuenchit
authors.initials: C
authors.lastname: Boonchird
authors.forename: Anocha
authors.initials: A
authors.lastname: Luengchaichawange
authors.forename: Pannipa
authors.initials: P
authors.lastname: Prompiboon
authors.forename: Jean
authors.initials: J
authors.lastname: Petre
authors.forename: Watanalai
authors.initials: W
authors.lastname: Panbangred
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics*; D016466:CHO Cells; D006224:Cricetinae; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin / Q000235:genetics*; D010567:Pertussis Vaccine / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D010567:Pertussis Vaccine; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; C068929:pertactin; D037342:Pertussis Toxin