Acellular antibordetella vaccine

Bacterial extract containing virulence factors of Bordetella, which is capable of participating in vaccinating compositions, as is obtained from phase I strains of B. pertussis, B. parapertussis or B. bronchiseptica by carrying out the following steps:

• Culture of the bacteria on solid blood Bordet-Gengou medium (BBG) for 48 hours so as to obtain isolated hemolytic colonies.
• Resuspension of a few colonies in liquid Stainer medium and plating out of 100 ml of this suspension on solid Stainer medium (CSM).
• After 24-36 hours of culture at 36° C., resuspension of the bacteria in liquid Stainer medium and inoculation of a 2-1 flask containing 200 ml of liquid Stainer medium such that the optical density at 650 nm is 0.05.
• After inoculation, culture for 18-20 hours at 36° C. with gentle agitation so as to obtain an optical density at 650 nm of 1-1.2,
• Centrifugation of the bacterial suspension for 30 minutes at 5000 g at 4° C.
• Resuspension of the bacterial pellet in 5M urea prepared in PBS buffer (described later) in the proportion of a volume equal to 5 times the wet weight of the bacterial pellet.
• Left stirring for 1 hour at 4° C., then centrifugation for 40 minutes at 40000 g at 4° C.,
• Storage of the supernatant at −80° C. until use
• Assay of the adenyl cyclase activity of the bacterial suspension thereby obtained without dilution. If this activity is between 30 and 100 U/ml, the urea extract may be prepared.

Bacterial extract containing expression products of the vrg genes, which is capable of participating in vaccinating compositions, as is obtained from phase IV strains of B. pertussis, B. parapertussis or B. bronchiseptica by carrying out the following steps:

• Culture of the bacteria on solid blood Bordet-Gengou medium (BBG) for 48 hours so as to obtain isolated hemolytic colonies.
• Resuspension of a few colonies in liquid Stainer medium and plating out of 100 ml of this suspension on solid Stainer medium (CSM).
• After 24-36 hours of culture at 36° C., resuspension of the bacteria in liquid Stainer medium and inoculation of a 2-1 flask containing 200 ml of liquid Stainer medium such that the optical density at 650 nm is 0.05.
• After inoculation, culture for 18-20 hours at 36° C. with gentle agitation so as to obtain an optical density at 650 nm of 1-1.2.
• Assay of the adenyl cyclase activity of the bacterial suspension thereby obtained without dilution. If this activity is between 30 and 100 U/ml, the urea extract may be prepared.