Industrial production method for Staphylococcus aureus vaccine

An industrial method of producing a vaccine comprising X-ray irradiated Staphylococcus aureus, the method comprising the following steps:

• (a) culturing Staphylococcus aureus bacteria of the ATCC 25923 strain on a tryptic soy agar (TSA) plate and then culturing in a tryptic soy broth (TSB) medium to obtain a primary seed,
• (b) inoculating the primary seed of step (a) into a TSB medium, adjusting the concentration to 0.01-0.1 optical density (OD) per ml and then performing culture expansion until the growth of the Staphylococcus aureus bacteria reaches logarithmic phase to obtain a secondary seed liquid,
• (c) inoculating the secondary seed liquid into a fresh TSB medium, adjusting the concentration to 0.01-0.1 OD per ml and performing culture expansion until the growth of the Staphylococcus aureus bacteria from the secondary seed liquid of step (b) reaches logarithmic phase to obtain a tertiary seed liquid,
• wherein final concentration of the seed liquid at each of the culture expansions is 0.8±0.2 OD/ml,
• (d) inoculating the tertiary seed liquid of step (c) into a fermentation tank containing a volume of TSB fermentation broth that is ⅓ to ½ of the total volume of the fermentation tank to produce a solution of the Staphylococcus aureus bacteria,
• (e) monitoring the concentration of the solution of the Staphylococcus aureus bacteria in the fermentation tank of step (d),
• (f) harvesting the solution from step (e) when the growth of the Staphylococcus aureus bacteria reaches logarithmic phase followed by directly centrifuging the solution with a centrifugal force of 2000 to 4000×g and collecting the centrifuged the Staphylococcus aureus bacteria after 10-30 minutes,
• (g) resuspending the Staphylococcus aureus bacteria collected in step (f) by isotonic injection and adjusting the concentration to 0.5-1×1010 of the bacteria per ml and then
• (h) performing intermittent X-ray irradiation of the resuspended Staphylococcus aureus bacteria at an irradiation dose rate of 5-20 Gy/minute for a total inactivating irradiation dose of 2000-3000 Gy with an interval of 5-10 minutes between the irradiations to cause the cells of the Staphylococcus aureus bacteria to lose their proliferative activity;
• and
• (i) adjusting the concentration of the X-ray irradiated Staphylococcus aureus bacteria to 0.5-1×108 per ml to obtain the X-ray irradiated Staphylococcus aureus vaccine,
• wherein the vaccine comprises whole cells and cell fragments, nucleic acids, and membrane vesicles of the Staphylococcus aureus strain released by the X-ray irradiation and wherein the vaccine has the capacity to confer protection against Staphylococcus aureus airway infection as determined in a mouse model of Staphylococcus aureus pneumonia or against Staphylococcus aureus blood infection as determined in a mouse model of Staphylococcus aureus bloodstream infection upon subcutaneous immunization of mice with 1 to 2×107 of the bacteria per ml dose of the X-ray irradiated Staphylococcus aureus vaccine.