Characterization of Polysaccharide Production of Haemophilus influenzae Type b and Its Relationship to Bacterial Cell Growth

Abstract

Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.

Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

“…Briefly, clinical isolates of Hib were sourced from National Institute of Communicable Diseases, Johannesburg, South Africa, and evaluated for PRP production in Frantz and CY media. Fermentation was performed at a 20 L scale using modified soy-peptone and yeast-extract (MP) medium [17] at 37°C, pH 7.2 with 15 g/L glucose addition, aeration (1.8 L/min) and stirring (300 rpm). After 15 h the OD 550 was in the range of 8-9, and PRP concentration was 800 to 1,200 mg/ L. Purification using a single step CTAB precipitation with Celite 545 was performed as described by Livyens [18].…”

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

“…inXuenzae type b GB 3291 was obtained from the Center for Disease Control and Prevention (Atlanta, GA), and the manufacturing master seed was prepared in the Department of Bacteriology at the Instituto Adolfo Lutz (São Paulo, SP, Brazil) [15,16].…”

“…The air supply was provided as air sparged aeration and maintained at 0.25 vvm (volume of air per volume of medium per minute). The pH was controlled at 7.4 and dissolved oxygen tension at 30% of air saturation, as described in Takagi et al [15,16].…”

“…Analytical procedures PRP concentration was measured by the modiWed Bial method, using ribose as standard [17] after dialysis of the samples [15,16]. The PRP concentration was determined by multiplying the ribose concentration by 2.55, according to the PRP structural formula reported by Crisel [18].…”

“…The pH of the supernatant EtOH 30% fraction was adjusted pH = 5.8, Na-acetate increased to 7% (w/v) and cold ethanol was added up to 80%, and processed as described above. The precipitated was resuspended in water and insoluble materials removed by centrifugation [15,16], water soluble EtOH 80% fraction.…”